Method using a nonlinear optical technique for detection of interactions involving a conformational change

ABSTRACT

A nonlinear optical technique, such as second or third harmonic or sum or difference frequency generation, is used to detect binding interactions, or the degree or extent of binding, that comprise a conformational change. In one aspect of the present invention, the nonlinear optical technique detects a conformational change in a probe due to target binding. In another aspect of the invention, the nonlinear optical technique screens candidate probes by detecting a conformational change due to a probe-target interaction. In another aspect of the invention, the nonlinear optical technique screens candidate modulators of a probe-target interaction by detecting a conformational change in the presence of the modulator.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of internationalapplication number PCT/US01/2241 1, entitled “Method and Apparatus Usinga Surface-Selective Nonlinear Optical Technique for Detection ofProbe-Target Interactions”, filed Jul. 17, 2001, which in turn claimsbenefit of U.S. provisional applications No. 60/253,862, entitled“Method and Apparatus Using a Surface-Selective Nonlinear OpticalTechnique for Detection of Probe-Target Interactions”, filed Nov. 29,2000; 60/260,249, entitled “Apparatus and Method for the Detection ofBiological Reactions Using a Surface-Selective Nonlinear OpticalTechnique”, filed Jan. 8, 2001; 60/265,775, entitled “Apparatus andMethod for the Detection of Biological Reactions Using aSurface-Selective Nonlinear Optical Technique”, filed Feb. 1, 2001; and60/278,941, entitled “Apparatus and Method for the Detection ofBiological Reactions Using a Surface-Selective Nonlinear OpticalTechnique”, filed Mar. 27, 2001 (accorded a filing date of Jan. 27,2001), the sum and substance of such applications being incorporated byreference herein in their entireties.

[0002] This application claims benefit of international applicationnumber PCT/US01/22441, entitled “Method and Apparatus Using aSurface-Selective Nonlinear Optical Technique for Detection ofProbe-Target Interactions Without Labels”, filed Jul. 17, 2001, which inturn claims benefit of U.S. provisional applications No. 60/260,261,entitled “Method and Apparatus Using a Surface-Selective NonlinearOptical Technique for Detection of Probe-Target Interactions WithoutLabels”, filed Jan. 8, 2001; 60/260,300, entitled “Apparatus and Methodfor the Detection of Biological Reactions Using a Surface-SelectiveNonlinear Optical Technique and an Indicator”, filed Jan. 8, 2001;60/262,214, entitled “Method and Apparatus Using a Surface-SelectiveNonlinear Optical Technique for Detection of Probe-Target InteractionsWithout Labels”, filed Jan. 17, 2001, the sum and substance of suchapplications being incorporated by reference herein in their entireties.

[0003] This application claims benefit of U.S. provisional applicationsNo. 60/306,040, entitled “Method and Apparatus Using a Nonlinear OpticalTechnique for Detection of Probe-Target Interactions in HomogeneousPhase Using an Applied Electric Field”, filed on Jul. 17, 2001;60/347,821, entitled “Method and Apparatus Using a Nonlinear OpticalTechnique for Detection of Probe-Target Interactions in an AppliedElectric Field”, filed on Oct. 23, 2001; 60/350,322, entitled “MethodUsing a Surface-Selective Nonlinear Optical Technique for Detection ofProbe-Target Interactions Using Molecular Beacons”, filed on Jan. 17,2002; 60/351,879, entitled “Method and Using a Surface-SelectiveNonlinear Optical Technique for Detection of Probe-TargetInteractions-Conformational Changes in The Probe”, filed on Jan. 24,2002; 60/354,668, entitled “Method and Apparatus Using a NonlinearOptical Technique for Detection of Probe-Target Interactions in anApplied Electric Field”, filed on Feb. 6, 2002; 60/354,679, entitled“Method and Using a Surface-Selective Nonlinear Optical Technique forDetection of Probe-Target Interactions-Conformational Changes”, filed onFeb. 6, 2002; 60/362,003, entitled “Method Using a Surface-SelectiveNonlinear Optical Technique for Detection of Interactions Involving aConformational Change”, filed on Mar. 5, 2002, which are herebyincorporated by reference in their entireties, including drawings.

1. FIELD OF THE INVENTION

[0004] The present invention relates to a method for detectinginteractions between biological components using a surface-selectivenonlinear optical technique. In one aspect of the present invention thisrelates to detection of binding between probes and targets that resultsin a conformational change.

2. BACKGROUND OF THE INVENTION

[0005] Detection of Binding

[0006] Detecting and quantifying interactions such as binding betweenbiomolecules is of central interest in modem molecular biology andmedicine. Ligand and drug binding to molecules is an important theme inmodem biology, medicine and drug development. Binding reactions betweenbiological components often result in a conformational or dipole moment(or induced dipole moment) change. Conformational change (a change inorientation or position of a molecule or subpart(s) of a molecule) is anessential feature of signaling in many systems. A technique fordetecting activation of a signal transducer (e.g., a receptor in amembrane), through a direct measure of conformational change or changein dipole moment, would be of value in basic research and drugdiscovery. For example, high-throughput drug screening for potentialagonists or antagonists of a receptor can be carried out using thepresent invention as a method for detection of whether potentialagonists or antagonists induce a conformational change—indicative ofbinding and activation of a receptor. Because the conformational changeis a direct indicator of receptor activation, it is an excellent meansof screening for drugs; current techniques often rely on indirectmeasures of activation such as changes in fluorescence intensity thatare concomitant with changes in Ca²⁺ ion concentration, which in turn iscaused by the receptor activation. Current techniques that directlymeasure activation, such as patch-clamping techniques with an ionchannel protein, are not amenable to high-throughput scaleup and requirea skilled technician to operate, resulting in higher cost to the user.

[0007] Fluorescence-based or surface plasmon-based detection are used todetect binding interactions, with varying success and efficiency.Problems using fluorescence include the presence of a naturalfluorescent background in many (non-labeled) biological samples as wellas photobleaching. Detection of orientational changes accompanyingtarget binding is difficult to do using fluorescence as the technique isnot very sensitive to label orientation: fluorescence polarization, notbeing a coherent technique, is sensitive to rotational motion during thefluorescence lifetime that makes it difficult to assign small measuredchanges in a particular polarization direction to conformational changes(rather than due to rotational motion). It is also difficult to separatesmall changes in probe orientation from the large fluorescent backgroundthat may be present in many biological samples. Furthermore, it is oftennot trivial to separate a signal indicative of binding from oneindicative of a receptor activation using fluorescence; for example,fluorescently tagged targets are tested for their ability to bind to agiven receptor using fluorescence polarization—but binding of a targetto a surface such as a cell surface can occur non-specifically such thatthe target does not specifically bind to the receptor of interest, yetcan still give a signal indicative of said binding; direct detection ofconformational change as an indicator of receptor activation is a moredirect means of assaying for activation. Wavelength-based changes due tochanges in the microenvironment of the label as a result ofconformational change can be followed, but not all conformationalchanges lead to a change in microenvironment and it may be difficult toassign relative changes due to microenvironement to the degree ofconformational change that actually occurs. Examples offluorescent-based prior art to detect conformational changes in proteinsinclude the following: Mannuzzu et al. (1996) Science 271, 213-216describe the direct physical measure of conformational rearrangementunderlying potassium channel gating using an fluorescently-taggedchannel protein. Gether et al., “Fluorescent labeling of purified β2adrenergic receptor,” J. Biol. Chem. 270, 28628-28275 (1995), describethe fluorescent labeling of soluble purified β2 adrenergic receptor todetect ligand-specific conformational changes. Turcatti et al., “Probingthe Structure and Function of the Tachykinin Neurokinin-Receptor throughBiosynthetic incorporation of flourescent amino acids at specificsites,” J. Biol. Chem. 271, 19991-19998, (1996), describe theincorporation of non-natural fluorescent amino acids into a receptor tomonitor ligand binding. Liu et al., “Site-Directed fluorescent labelingof P-glycoprotein on cysteine residues in the nucleotide bindingdomains,” Biochemistry 35, 11865-11873, (1996), describe fluorescentlabeling of soluble purified P-glycoprotein to detect ligand binding.

[0008] Fluorescence has also been used to detect binding of targets toion channel receptors when the binding leads to a change intransmembrane potential in cells. The transmembrane potential change(e.g., a depolarization) leads to a change in the intensity, lifetime,wavelength, etc. of the fluorescent or nonlinear-active label in themembrane. Apart from various problems in the detection itself—concerningphotobleaching, artifacts and background noise, these methods onlyprovide indirect assays for the probe-target binding. For example, it ispossible that a given target binds to ion channel probes and the ionchannels are activated, but that this does not lead to a change intransmembrane potential, either for natural reasons or because there areproblems in the cells in the normal mechanism for producing the changein transmembrane potential. It is desirable therefore to have a direct,optical means of detecting probe-target binding reactions in cases wherethe binding reaction results in a change in orientation or conformationof the probe.

[0009] Molecular Beacons

[0010] Molecular beacons are also used for the detection of bindinginteractions. A molecular beacon (MB) probe is well known in the art asa hairpin-loop, single-stranded oligonucleotide comprising a probesequence embedded within complementary sequences that form a hairpinstem. The loop portion of the molecule can form a double-stranded DNA inthe presence of complementary nucleic acid. A fluorophore is covalentlyattached to one end of the oligonucleotide, and a nonfluorescentquencher is covalently attached to the other end. There are typicallyfive to eight bases at each side of the two ends of the beacon which arecomplementary to each other. The stem keeps these two moieties in closeproximity to each other, causing the fluorescence of the fluorophore tobe quenched by energy transfer. When the beacon binds to its target, therigidity of the probe-target duplex forces the stem to unwind, whichcauses the separation of the fluorophore and the quencher and therestoration of fluorescence. This permits the detection of probe-targethybrids in the presence of unhybridized probes.

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[0104] Strouse R J, Hakki F Z, Wang S C, DeFusco A W, Garrett J L, andSchenerman M A (2000) Using molecular beacons to quantify low levels oftype I endonuclease activity. Biopharm 13, 40-47.

[0105] Tan W, Fang X, Li J, and Liu X (2000) Molecular beacons: a novelDNA probe for nucleic acid and protein studies. Chemistry 6, 1107-1111.

[0106] Thelwell N, Millington S, Solinas A, Booth J, and Brown T (2000)Mode of action and application of scorpion primers to mutationdetection. Nucleic Acids Res 28, 3752-3761.

[0107] Tung C H, Mahmood U, Bredow S, and Weissleder R (2000) In vivoimaging of proteolytic enzyme activity using a novel molecular reporter.Cancer Res 60, 4953-4958.

[0108] Whitcombe D, Theaker J, Guy S P, Brown T, and Little S (1999)Detection of PCR products using self-probing amplicons and fluorescence.Nat Biotechnol 17, 804-807.

[0109] Yamamoto R, Baba T, and Kumar P K (2000) Molecular beacon aptamerfluoresces in the presence of Tat protein of HIV-1. Genes Cells 5,389-396.

[0110] Ying L, Wallace M I, and Klenerman D (2001) Two-state model ofconformational fluctuation in a DNA hairpin-loop. Chemical PhysicsLetters 334, 145-150.

[0111] Zhang P, Beck T, and Tan W (2001) Design of a molecular beaconDNA probe with two fluorophores. Angew. Chem. Int. Ed. 40, 402-405.

[0112] Nonlinear Optical Techniques

[0113] Surface-selective nonlinear optical techniques have previouslybeen confined mainly to physics and chemistry since relatively fewbiological samples are intrinsically non-linearly active. Examplesinclude the use of an optically nonlinear-active dye as a membrane stainand an endogenous nonlinear-active stain (GFP) that is used to imagebiological cells (Campagnola et al., “High-resolution nonlinear opticalimaging of live cells by second harmonic generation,” BiophysicalJournal 77 (6), 3341-3349 (1999), Peleg et al., “Nonlinear opticalmeasurement of membrane potential around single molecules at selectedcellular sites,” Proc. Natl. Acad. Sci. V. 96, (1999), 6700-6704 andreferences therein). The following references (and references therein)are exemplary of this art:

[0114] A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N.Friedman, O. Bouevitch, Z. Rothman, L. Loew and M. Sheves, “SecondHarmonic Generation of Biological Interfaces: Probing the MembraneProtein Bacteriorhodopsin and Imaging Membrane Potential Around GFPMolecules at Specific Sites in Neuronal Cells of C. elegans,” ChemicalPhysics 245, 133 (1999).

[0115] O. Bouevich, A. Lewis, I. Pinnevsky and L. Loew, “ProbingMembrane Potential with Non-linear Optics,” Biophys. J. 65, 672 (1993).

[0116] I. Ben-Oren, G. Peleg, A. Lewis, B. Minke and L. Loew, “Infrarednonlinear optical measurements of membrane potential in photoreceptorcells,” Biophys. J. 71, 616 (1996).

[0117] G. Peleg, A. Lewis, M. Linial and L. M. Loew, “Non-linear OpticalMeasurement of Membrane Potential Around Single Molecules at SelectedCellular Sites,” Proc. Acad. Sci. USA 96, 6700 (1999).

[0118] P. Campagnola, Mei-de Wei, A. Lewis and L. Loew, “High-ResolutionNonlinear Optical Imaging of Live Cells by Second Harmonic Generation,”Biophys. J. 77, 3341 (1999).

[0119] J. Y. Huang, A. Lewis and L. Loew, “N[on-linear OpticalProperties of Potential Sensitive Styryl Dyes”, Biophysical J. 53, 665(1988).

[0120] A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N.Friedman, O. Bouevitch, Z. Rothman, L. Loew and M. Sheves, “SecondHarmonic Generation of Biological Interfaces: Probing Membrane Proteinsand Imaging Membrane Potential Around GFP Molecules at Specific Sites inNeuronal Cells of C. elegans,” Chemical Physics 245, 133-144(1999).

[0121] A. Khatchatouriants, A. Lewis, Z. Rothman, L. Loew and M.Treinin, “GFP is a Selective Non-Linear Optical Sensor ofElectrophysiological Processes in C. elegans,” Biophys. J. (in press,2000)

[0122] In the prior art, nonlinear-active stains are immobilized inmembranes and these stains are used to image the cell surfaces. However,the stains intercalate into the membranes in either an ‘up’ or ‘down’direction, thus reducing the total nonlinear signal due to destructiveinterference. Nonlinear optically active dyes have also been used tomeasure the kinetics of those dyes crossing lipid bilayers in liposomes(A. Srivastava and K B Eisenthal, “Kinetics of molecular transportacross a liposome bilayer,” Chem. Phys. Lett. 292 (3): 345-351 (1998) ).

[0123] The present invention provides advantages over fluorescence-baseddetection, such as reduced background, reduced photobleaching,simplified optical detection, and no need for labor- or time-consumingwashing steps. These advantages are due to the low background of thenonlinear optical techniques and the fact that the method is ascattering rather than an absorption process.

[0124] It is therefore an object of the present invention to provide adirect, optical means of detecting probe-target binding reactions incases where the binding reaction results in a change in orientation orconformation of the probe, target, or both probe and target.

3. SUMMARY OF THE INVENTION

[0125] The invention discloses a method for screening one or morecandidate binding partners (referred to herein as probes) for binding toa test molecule (referred to herein as targets). The method involvesilluminating a sample with one or more light beams at one or morefundamental frequencies, said sample comprising the test moleculeexposed to the one or more candidate binding partners, and measuring oneor more physical properties of a nonlinear optical light beam emanatingfrom the sample. A change in the value of the one or more physicalproperties measured relative to a value for the one or more physicalproperties measured in the absence of exposure of said test molecule tosaid one or more candidate binding partners indicates that said one ormore candidate binding partners bind said test molecule.

[0126] The invention discloses a method for screening one or morecandidate modulator molecules for the ability to modulate an interactionbetween a test molecule and its binding partner. The method involvesilluminating a sample with one or more light beams at one or morefundamental frequencies, said sample comprising said test moleculeexposed to (i) said binding partner, and (ii) said one or more candidatemodulator molecules, and measuring one or more physical properties of anonlinear optical light beam emanating from said sample. A change in thevalue of said one or more physical properties measured relative to thevalue for said one or more physical properties measured in the absenceof exposure to said one or more candidate modulator molecules indicatesthat said one or more candidate modulator molecules modulate theinteraction between said test molecule and its binding partner.

[0127] The invention provides a method for detecting a conformationalchange in a test molecule upon binding of the test molecule to a bindingpartner comprising contacting said test molecule with one or morecandidate binding partners, where the test molecule or the one or morecandidate binding partners is labeled with a nonlinear-active moietythat is not native to the test molecule or the one or more candidatebinding partners, respectively. The method involves illuminating saidcontacted test molecule with one or more light beams at one or morefundamental frequencies, and measuring one or more physical propertiesof a nonlinear optical light beam emanating from said sample. A changein the value of said one or more physical properties measured relativeto the value for said one or more physical properties measured in theabsence of said one or more candidate binding partners indicates that atleast one of said one or more candidate binding partners bind to saidtest molecule and that said binding induces a conformational change insaid candidate binding partners, in said test molecule, or in both saidcandidate binding partners and said test molecule.

[0128] The invention provides a method for detecting the degree orextent of the binding interaction between a test molecule and one ormore binding partner comprising contacting said test molecule with oneor more candidate binding partners, wherein the test molecule or the oneor more candidate binding partners is labeled with a nonlinear-activemoiety that is not native to the test molecule or the one or morecandidate binding partners, respectively. The method involvesilluminating said contacted test molecule with one or more light beamsat one or more fundamental frequencies, and measuring one or morephysical properties of a nonlinear optical light beam emanating from thesample. A change in the value of said one or more physical propertiesmeasured relative to the value for the one or more physical propertiesmeasured in the absence of said one or more candidate binding partnersindicates that at least one of said one or more candidate bindingpartners binds to said test molecule, the degree or extent of theconformational change that said binding induces.

[0129] In a specific embodiment, the present invention relates to amethod for detecting interactions between biological components using asurface-selective nonlinear optical technique. In one aspect of thepresent invention this relates to detection of binding between probesand targets that results in a conformational change. The inventiondiscloses methods for screening one or more probes through theconformational changes they induce on binding targets. The inventionalso discloses methods for screening modulators of the probe-targetbinding interaction, and for determining the degree or extent of bindingthrough the conformational changes the binding induces.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0130]FIG. 1 depicts one embodiment of the apparatus in which the modeof generation and collection of the second harmonic light is byreflection off the substrate with surface-attached probes.

[0131]FIG. 2 depicts one embodiment of an apparatus in which the mode ofgeneration and collection of the second harmonic light is by totalinternal reflection through a prism. The prism is coupled by anindex-matching material to a substrate with surface-attached probes.

[0132]FIG. 3 depicts one embodiment of an apparatus in which the mode ofgeneration and collection of the second harmonic light is by totalinternal reflection through a wave-guide with multiple reflections asdenoted by the dashed line inside the wave-guide.

[0133] FIGS. 4A-D depict one embodiment of a flow-cell for delivery andremoval of biological components and other fluids to the substratecontaining attached probes.

[0134] FIGS. 5A-C depict three embodiments of an apparatus in which themode of generation and collection of the second harmonic light is bytransmission through a sample. In FIG. 5A, the second harmonic beam isco-linear with the fundamental. In FIG. 5B, the second harmonic iscollected from a direction orthogonal to the fundamental (‘right-anglecollection’). In FIG. 5C, the second harmonic light is collected by anintegrating sphere and a fiber optic line.

[0135]FIG. 6 depicts an embodiment of the transformation, using a seriesof optical components, of a collimated beam of the fundamental lightinto a line shape suitable for scanning a substrate.

[0136] FIGS. 7A-B depict an embodiments patterned in an array format.FIG. 7A depicts an embodiment of a substrate surface (containingattached probes) which has been patterned into an array format (elements1-35). FIG. 7B depicts one element of a substrate array in which eachelement is a well with walls, with surface-attached probes, and the wellis capable of holding some liquid and serves to physically separate thewell's contents from adjacent wells or other parts of the substrate.

[0137]FIG. 8 depicts one embodiment of a surface chemistry used toattach oligonucleotide or polynucleotide samples to the substratesurface.

[0138] FIGS. 9A-B depict an embodiment of a substrate containingmultiple wells. FIG. 9A depicts the substrate containing multiple wells(1-16), each of which contains surface-attached probes as depicted inFIG. 9B.

[0139]FIG. 10 depicts an embodiment of the apparatus substrate with theuse of an aminosilane surface-attached layer on top of a reflectivecoating. The reflective coating underneath the aminosilane layerimproves collection of the nonlinear optical light. The aminosilanelayer is suitable for coupling biomolecules or other probe components tothe substrate.

[0140] FIGS. 11A-B depict an embodiment of an apparatus in which themode of generation and collection of the second harmonic light isthrough a fiber optic. FIG. 11 A depicts the use of a bundle of fiberoptic lines and FIG. 11B depicts the use of beads coupled to the end ofa fiber for attaching probes.

[0141] FIGS. 12A-C depict three embodiments of an apparatus in which themode of generation and collection of the second harmonic light is bytransmission through a sample. FIG. 12A depicts both the fundamental andsecond harmonic beams travelling co-linearly through a sample. FIG. 12Bdepicts the fundamental and second harmonic beams being refracted at thetop surface (top surface contains attached probes) of a substrate withthis surface generating the second harmonic light. FIG. 12C depicts asimilar apparatus to FIG. 12B except that the bottom surface (bottomsurface contains attached probes) generates the second harmonic light.

[0142] FIGS. 13A-B depict two embodiments of an apparatus in whichsecond harmonic light is generated by total internal reflection at aninterface. The points of generation of the second harmonic light aredenoted by the circles. In FIG. 13A, a dove prism is used to guide thelight to a surface capable of generating the second harmonic light(bottom surface of prism but can also be another surface coupled to theprism through an index-matching material). In FIG. 13B, a wave-guidestructure is used to produce multiple points of second harmonicgeneration.

[0143] FIGS. 14A-C depict three embodiments of an apparatus in whichsecond harmonic light is generated using a fiber optic line (withattached probes at the end of the fiber). FIG. 14A depicts an apparatusin which both generation and collection of the second harmonic lightoccur in the same fiber. FIG. 14B depicts the use of a bead containingsurface-attached probes at the end of the fiber. FIG. 14C depicts anapparatus in which the second harmonic light is generated at the end ofthe fiber optic (containing attached probges) and collected using amirror or lens external to the fiber optic.

[0144] FIGS. 15A-B depict two embodiments of an apparatus using anoptical cavity for power build-up of the fundamental.

[0145] FIGS. 16A-C depict three embodiments of an apparatus in which themode of generation and collection of the second harmonic light usesreflection of the light from an interface.

[0146]FIG. 17 depicts one embodiment of the sample cell with an appliedelectric field. The sample cell (5) is open and is depicted side-on andfilled with solution (10) containing probes and targets. The fundamentalbeam enters the sample cell to the right (15) and passes throughtransparent electrodes (20) separated by a spacer of 1-3 mm (25). Theelectrodes are connected to a source of voltage (30). When voltage isapplied, the targets and probes become partially oriented, thusproducing the nonlinear optical light (e.g., second harmonic light).

[0147]FIG. 18 depicts an apparatus for measuring probe-targetinteractions in suspended cells. A source of fundamental light from aTi:Sapphire femtosecond laser (1000) operating at 800 nm is directedthrough a filter (RG-645, CVI Laser) that blocks 400 nm light (secondharmonic). The fundamental is then focused using plano-convex lens 1200(100 mm focal length, Melles Griot) into sample holder 1300 containing asuspension of biological cells. The second harmonic light is collectedparallel to the fundamental direction using a 80 mm diameterplano-convex collimating lens (1400) (120 mm focal length, Melles Griot)that is positioned to have its focus at the focus of lens 1200. Thelight is sent through a color filter (BG-39, CVI Laser) to block thefundamental light and pass second harmonic light on to a secondplano-convex lens 1600 (120 mm focal length) Melles Griot which focusesthe light through a monochromator slit and onto its grating (1700). Aphotomultiplier is attached to the monochromator and detects the secondharmonic light. The signal from the photomultiplier is sent on to photoncounting electronics (SR400, Stanford Research Systems) and then passedto a computer 1900 for storage and analysis of the data.

[0148] FIGS. 19A-C depict various embodiments of the present invention.In 19A, receptors embedded in a membrane surface are exposed to ligandsor particles for which they have an affinity. After the bindinginteraction occurs, the ligands or particles are bound to themembrane-associated receptors. In 19B, cells are attached (or ‘plated’)to a surface or substrate to form approximately a monolayer of cells(e.g., ˜100% confluent cells); cells can also be stacked to formmultilayers at a substrate or surface. In both cases in 19B, incidentfundamental light can be transmitted through the substrate or surface,run parallel to the surface and through the multilayer or monolayer ofcells, coupled to the cell layer evanescently, or some combinationthereof. FIG. 19C depicts a conformational change process. Receptors ina membrane surface (e.g., a host cell) are labeled with nonlinear-activelabels which, on average, possess an orientation with respect to thesurface plane, specifically an angle of θ of the hyperpolarizabilitywith respect to the surface plane; binding and subsequent activation ofthe receptor by ligands causes the labels to shift orientation to angleβ. Small shifts in angle can cause a substantial change in a physicalproperty of the measured nonlinear optical light (e.g., intensity of thelight).

[0149] FIGS. 20A-B depict a molecular beacon that has been modified toform a nonlinear-active analogue. In FIG. 20A, a single strand ofnucleotides is coupled to a nonlinear-active dye (gray-hatch) and anenhancer (open circle). When the molecular beacon analogue is hybridizedto a complementary target, the dye and enhancer are separated by theconformational change, leading to a measurable change in the nonlinearresponse of the dye. The amount of the change can be made quantitativewith the amount of probe-target hybridization. In FIG. 20B, an exampleof a particular oligonucleotide sequence with attached dye and enhancer(Au particle) is shown (SEQ ID NO: 5). The sequences of various targets(SEQ ID NOs: 1-4, respectively) used for testing the degree ofhybridization to this probe are also shown.

5. DESCRIPTION OF THE INVENTION

[0150] The present invention uses a nonlinear optical technique todetect probe-target interactions involving a conformational change.Examples of nonlinear optical technique include but are not limited tosecond-harmonic generation, sum-frequency generation,difference-frequency generation, third-harmonic generation andhyper-Rayleigh scattering HRS). The present invention can be used withany of the nonlinear optical techniques, however many embodimentsdescribe the use of the second-order techniques (e.g., second-harmonicgeneration and hyper-Rayleigh scattering). The following references, andreferences therein, describe the field of nonlinear optics:

[0151] Nonlinear Optics, R. W. Boyd, 1991, Academic Press.

[0152] The Elements of Nonlinear Optics, P. N. Butcher and D. Cotter,Cambridge University Press, 1991.

[0153] Nonlinear Optical Techniques

[0154] Nonlinear optical light is any light that results from anonlinear transformation of light beams at one or more fundamentalfrequencies (also referred to herein as fundamental beam(s)). Anonlinear optical technique is capable of transforming the physicalproperties, such as frequency, intensity, etc., of one or more incidentlight beams, called the fundamental beams. The nonlinear beams emanatingfrom the sample are the higher order frequency beams, e.g. second orthird harmonic, etc., or the beams at the sum or difference frequencies.For example, in second harmonic generation (SHG), two photons of thefundamental beam are virtually scattered by the sample to produce onephoton of the second harmonic. A nonlinear optical technique is alsoreferred to herein as a surface-selective nonlinear optical technique.

[0155] Second harmonic generation (SHG) and other surface-selectivenonlinear optical techniques are directly related to the orientation ofthe nonlinear-active species in a sample, because the fundamental andnonlinear beams have well-defined phase relationships, and thewavefronts of the nonlinear beam in a macroscopic sample (within thecoherence length) are in phase. Any change in the orientation of thenonlinear-active species can be detected by measuring one or morephysical properties of the nonlinear optical beam emanating from thesample. These coherency properties of the nonlinear optical techniqueoffer a number of advantages useful for surface or high-throughputstudies in which, for example, either a single surface or a microarraysurface is examined. The coherent nature of the nonlinear optical beamemanating from the sample also allows discrimination among more than onenonlinear optical beam emanating from a sample. In alternate embodimentsof the invention, assays can be conducted where multiple fundamentallight beams at one or more frequencies, incident with one or morepolarization directions relative to the sample, can be used, with theresulting emanation of at least two nonlinear beams. An apparatus usingnonlinear optical suface-selective-based detection, such as with secondharmonic generation, requires minimal collection optics since generationof the nonlinear light only occurs at the interface (in the absence ofan applied field) and thus, in principle, allows extremely high depthdiscrimination and fast scanning.

[0156] A common equation used to model orientation dependence ofnonlinear-active species at an interface is:

χ⁽²⁾ =Ns<α ⁽²⁾>

[0157] where χ⁽²⁾ is the nonlinear susceptiblity, N_(s) is the totalnumber of molecules per unit area at the interface and <α⁽²⁾> is theaverage over the orientational distribution of the nonlinearhyperpolarizabilities—α⁽²⁾—in these molecules. Typical equationsdescribing the nonlinear interaction for second harmonic generation are:α⁽²⁾(2ω)=β·E(ω)).E(ω) or P⁽²⁾(2ω)=χ⁽²⁾:E(ω)E(ω) where α and P are,respectively, the induced molecular and macroscopic dipoles oscillatingat frequency 2ω, β and χ⁽²⁾ are, respectively, the hyperpolarizabilityand second-harmonic (nonlinear) susceptibility tensors, and E(ω) is theelectric field component of the incident radiation oscillating atfrequency ω. The macroscopic nonlinear susceptibility χ⁽²⁾ is related byan orientational average of the microscopic β hyperpolarizability. Thenext order term in the expansion of the induced macroscopic dipoledescribes other nonlinear phenomenon, such as third harmonic generation.The third order term is responsible for such nonlinear phenomena astwo-photon fluorescence. For sum or difference frequency generation, thedriving electric fields (fundamentals) oscillate at differentfrequencies (i.e., ω₁ and ω₂) and the nonlinear radiation oscillates atthe sum or difference frequency (ω₁±ω₂).

[0158] The intensity of SHG is proportional to the square of thenonlinear susceptiblity and is dependent on the amount of orientednonlinear-active species in a sample, and thus to changes in thisorientation, both at an interface and species aligned in the bulk (thelatter through an electric field-poled mechanism, for example). Thisproperty can be exploited to detect a conformational change. Forexample, conformational change in receptors can be detected using anonlinear-active label or moiety wherein the label is attached to orassociated with the receptor; a conformational change leads to a changein the direction (orientation) of the label with respect to the surfaceplane (or applied field direction) and thus to a change in a physicalproperty of the nonlinear optical signal. The techniques areintrinsically sensitive to these changes at an interface and can be madesensitive to them in the bulk as well, by applying an electric field topole molecules or simply by detecting that fraction of the ensemblewhich produce hyper-Rayleigh scattering (HRS) due to fluctuationalchanges in their number density or orientation as is well known to oneskilled in the art.

[0159] In hyper-Rayleigh scattering (HRS), the fluctuations ofnonlinear-active molecules lead to instantaneous departures fromcentrosymmetry, and thus allow for a low amount of second-harmonicemission to occur, although this emission is incoherent. Because thefluctuations depend on molecular size, among other properties, HRS canbe used to discriminate an unbound molecule in solution from the samemolecule bound to one or more binding partners (also referred to hereinas probes). Thermal energy drives the fluctuations required for HRS;however, an external force can also be applied to induce or amplify thefluctuations, thus increasing the HRS signal. For example, a flow-fieldcan be used to transiently orient molecules in solution by injecting aburst or stream of fluid into it. Pulsed and alternating electric fieldsapplied to the sample can also increase the HRS signal.

[0160] There are a number of examples in the literature of the use ofHRS to measure beta (hyperpolarizability) of nonlinear opticalmolecules. The present invention extends the use of HRS to detectbinding interactions and to screen for test molecules (also referred toherein as targets) or modulators (described below) capable of binding ormodulating probe-target interactions.

[0161] The following references, and references therein, describe theHRS technique:

[0162] Clays, K. et al., “Nonlinear Optical Properties of ProteinsMeasured by Hyper-Rayleigh Scattering in Solution”, Science, v. 262(5138), 1419-22

[0163] Vance, F W, Lemon B. I., Hupp, J. T. Enormous Hyper-RayleighScattering from Nanocrystalline Gold Particle Suspensions”, J. Phys.Chem. B 102:10091-93 (1999).

[0164] Electric field induced second harmonic (EFISH) is technique wellknown in the field of nonlinear optics that can be used according to theinvention to render a system nonlinear-active that is not normally so,such as a bulk phase, through the application of an electric field thatbreaks the symmetry of the bulk phase. In a specific embodiment, theEFISH technique can be used to measure the hyperpolarizabilty ofmolecules in solution by using a dc field to induce alignment in themedium, and allowing nonlinear-activity (such as SHG) to be observed.This is sometimes called the reorientational mechanism.

[0165] EFISH is a third order nonlinear optical effect, with thepolarization source written as:

P ⁽²⁾(ω₃)=χ⁽²⁾(−ω₃; ω₁,ω₂):E ^(ω1) E ¹⁰⁷ ²

[0166] The polarization is the result of the application of two opticalfields and a static (dc) field. The following references describeapplying the EFISH technique to, e,g, liquid and condensed phasesamples:

[0167] R. Dworczak and D. Kieslinger, Phys. Chem. Chem. Phys., 2000, 2,5057-5064.

[0168] J. L. Oudar, J. Chem. Phys., 1977, 67, 446.

[0169] B. F. Levine and C. G. Bethea, J. Chem. Phys., 1974, 60, 3856.

[0170] B. F. Levine and C. G. Bethea, J. Chem. Phys., 1975, 63, 2666.

[0171] K. D. Singer and A. F. Garito, J. Chem. Phys., 1981, 75, 3572.

[0172] C. G. Bethea, Applied Optics, 1975, 14,1447.

[0173] B. F. Levine and C. G. Bethea, J. Chem. Phys., 1976, 65, 1989.

[0174] B. F. Levine and C. G. Bethea, J. Chem. Phys., 1974, 60, 3856.

[0175] B. F. Levine, J. Chem. Phys., 1975, 63, 115.

[0176] B. F. Levine and C. G. Bethea, J. Chem. Phys. 1977, 66, 1070.

[0177] J. L. Oudar and D. S. Chemla, J. Chem. Phys., 1977, 66, 2664.

[0178] R. S. Finn and J. F. Ward, J. Chem. Phys., 1974, 60, 454.

[0179] B. F. Levine and C. G. Bethea, Appl. Phys. Lett., 1974, 24, 445.

[0180] In addition, the electrodes used to apply the electric field canbe spatially patterned (periodically patterned) to achieve quasi-phasematching. Quasi-phase matching is a well known technique for increasingthe effective nonlinear path length in a nonlinear-active material, byalternating the nonlinear susceptibility of a material with a period ofa coherence length. The following references (and references therein)describe this technique:

[0181] M. Jäger et al., Appl. Phys. Lett. 68, 1996, 1183.

[0182] M. M. Fejer et al., IEEE Journal of Quantum Electronics, v. 28,1992, 2631.

[0183] G. D. Landry et T. A. Maldonado, Optics Express, v.5, 1999, 176.

[0184] The electrodes used to apply the external electric field in theEFISH technique can assume a variety of shapes, forms and compositionsas, for example, are found in the prior art. For instance, theelectrodes can be angled or pointed to increase the electric fieldstrength that results in these cases. The electrodes can be oriented ina variety of ways to the sample; for example, the electrodes can beplaced (e.g., patterned lithographically, printed, etched, etc.) on asubstrate which itself is in contact with the liquid sample containingthe targets and probes; or, for example, the electrodes can lie at thebottom and top of a thin cavity in which the sample containing targetsand probes is flowing or is held.

[0185] Non-Random Orientations and Nonlinear Activity

[0186] A non-random orientation is a necessary condition for generationof the surface-selective nonlinear optical signal. Only thenon-centrosymmetric region of a system, is capable of generatingnon-linear light. A molecule or material phase is centrosymmetric ifthere exists a point in space, called the ‘center’ or ‘inversioncenter,’ through which an inversion (x,y,z)→(−x,−y,−z) of all atoms canbe performed that leaves the molecule or material unchanged. Forexample, if the molecule is of uniform composition and spherical inshape, it is centrosymmetric. Centrosyrnmetric molecules or materialshave no nonlinear susceptibility or hyperpolarizability, necessary forsecond or higher harmonic, sum frequency and difference frequencygeneration. A non-centrosymmetric molecule or material lacks this centerof inversion, and therefore can be nonlinear-active. Non-centrosymmetricregions can be at surfaces, e.g. arrays, substrates, etc., or in bulkphase, e.g. solutions, however, a bulk phase may require the applicationof an electric field to break the symmetry of the region and render thebulk phase nonlinear-active (as in the EFISH technique).

[0187] The present invention exploits the property that any non-randomorientation of probes with respect to a surface to which they areattached or localized leads to non-random orientation of targets whenthese targets bind to the probes. As a result of the non-randomorientation of the system, a nonlinear optical technique can be used tomonitor, e.g., the binding activity at the surface. Any change in thenon-random orientation of the system, such as due to a conformationalchange on occurrence of a binding event, would modify the nonlinearoptical signal. Verification that a change in the detected nonlinearoptical signal is due to a conformational change can be accomplishedusing controls known to one skilled in the art including, for example,measuring binding of a blocking compound to the receptor—where theblocking compound is known not to produce a conformational change in thereceptor.

[0188] In an embodiment in which the probe or target, or neither, is notnatively nonlinear-active, then different types of nonlinear-activespecies can be introduced into the system to render the systemnonlinear-active. Such species that affect the one or morenonlinear-active beams emanating from a sample comprising probes andtargets include labels, decorators, indicators, modulators, inhibitors,and enhancers, as described below. When the present invention is usedwith labels or decorators, the non-random orientation of the targetproduces a non-random label or decorator orientation and this leadsdirectly to an increase in surface-selective nonlinear optical signal(e.g., intensity). Non-specific interactions of the targets withattached or localized probes (e.g., non-specific binding to probes) orof targets to regions on the surface where no probe is present (e.g.,non-specific binding to substrate or solid support) will lead to zero orto a much lower surface-selective nonlinear optical signal due todestructive interference as would be apparent to one skilled in the art.When the present invention is used with indicators, a non-random probeorientation also leads to an increase in the surface-selective nonlinearoptical signal since the surface charge density close to the surfaceplane will be larger than if the probes are randomly oriented whichresults in a lower surface electric charge density.

[0189] Nonlinear-Active Labels

[0190] A label for use in the present invention refers to anonlinear-active moiety, particle or molecule which can be bound, eithercovalently or non-covalently, to a molecule, particle or phase (e.g.,lipid bilayer) in order to render the resulting system more nonlinearoptical active. Labels can be employed in the case where the molecule,particle or phase (e.g., lipid bilayer) is not nonlinear-active torender the system nonlinear-active, or with a system that is alreadynonlinear-active to add an extra manipulation parameter into the system.The exogenous labels can be pre-attached to the molecules or particles,and any unbound or unreacted labels separated from the labeled entitiesbefore a measurement is made. In a specific embodiment, thenonlinear-active moiety is attached to the target or probe molecule invitro. Alternatively, the labels can be left in solution with probes andtargets and allowed to adsorb to some particle (e.g., an enhancer) orsurface to yield a different nonlinear-active response (i.e.,hyperpolarizability or second order susceptibility) from the boundlabels. By way of example, EFISH or Hyper-Rayleigh scattering can beused to determine if a candidate molecule or particle is nonlinearlyactive according to techniques well known in the art, where appropriatecontrols and background measurements are made in the absence of accessto the candidate nonlinear-active species. The labeling of probes withnonlinear-active labels and/or modulators of the labels allows a direct,optical means of detecting probe-target binding reactions in the caseswhere the binding reaction results in a change in orientation orconformation of the probe using a surface-selective nonlinear opticaltechnique.

[0191] In alternate embodiments of the invention, at least twodistinguishable nonlinear-active labels are used. The orientation of theattached two or more distinguishable labels would then be chosen tofacilitate well defined directions of the emanating coherent nonlinearlight beam. The two or more distinguishable labels can be used in assayswhere multiple fundamental light beams at one or more frequencies,incident with one or more polarization directions relative to thesample, are used, with the resulting emanation of at least two nonlinearlight beams.

[0192] One means of determining whether a particular molecule orparticle can be used as a nonlinear-active label is by studying it usingsecond harmonic generation at an air-water interface. For instance, inthe case of particles, if the particles assemble at the air-waterinterface in a manner which gives a net orientation of the particles (ona length scale of the coherence length) the layer of particles willgenerate second harmonic light. Another means of doing this is bymeasuring a sample of a suspension of the particles and detecting thehyper-rayleigh scattering. Yet another means involves the use of EFISHto determine if a candidate molecule or particle is nonlinearly active.The effect can be used to measure the hyperpolarizabilty of molecules insolution by using a de field to induce alignment in the medium, andallowing SHG to be observed. This type of measurement does not requirethat the particle themselves be ordered at an interface, but doesrequire that the particles be nonlinear-active and thusnon-centrosymmetric.

[0193] In a specific embodiment, metal nanoparticles and assembliesthereof are modified to create biological nonlinear-active labels, asdescribed in the fo. The following references describe the modificationmetal nanoparticles and assemblies:

[0194] J. P. Novak and D. L. Feldheim, “Assembly ofPhenylacetylene-Bridged Silver and Gold Nanoparticle Arrays”, J. Am.Chem. Soc., 2000, 122, 3979-3980.

[0195] J. P. Novak et al., “Nonlinear Optical Properties of MolecularlyBridged Gold Nanoparticle Arrays”, J. Am. Chem. Soc. 2000, 122,12029-12030.

[0196] Vance, F W, Lemon B. I., Hupp, J. T. Enormous Hyper-RayleighScattering from Nanocrystalline Gold Particle Suspensions”, J. Phys.Chem. B 102:10091-93 (1999).

[0197] The following reference and references therein describe thetechniques available for creating a biological label from a syntheticdye and many other molecules:

[0198] Greg T. Hermanson, Bioconjugate Techniques, Academic Press, 1996.

[0199] In a specific embodiment, nonlinear-active labels can beconstructed according to well known procedures in the art to bephotoactivated or photomodulated with a beam of light such that, uponirradiation of the sample with a selected beam of light, the labelsbecome nonlinear optical active (or more or less nonlinear opticalactive). The beam of light can, for example, cleave a chemical bond(e.g., using UV light), well known in the art as ‘caged’ compounds.

[0200] Modulators

[0201] Modulators include any substance (e.g., moiety, molecule,biological component or compound) that alters the nonlinear response ofa nonlinear-active species when the modulator is in proximity to thenonlinear-active species, or alters the kinetic or equilibriumproperties of probe-target interactions (e.g., binding reaction).Modulators may change the rate of probe-target binding, the equilibriumconstant of probe-target binding or, in general, enhance or reduceprobe-target interactions. Examples of modulators are the following:inhibitors, drugs, small molecules, agonists and antagonists and Auparticles. Candidate modulators can be tested for their ability toperform as modulators, e.g., in a screening process.

[0202] In a specific embodiment, target-probe interactions can bemeasured in the presence of some modulator of the interactions—themodulator being, for example, a small molecule, drug, or other moiety,molecule or particle which changes in some way the target-probeinteractions (e.g., has some affinity for the probe and blocks orinhibits target binding). The modulator can be added before, during orafter the time in which the probe-target interactions occur.

[0203] Inhibitors

[0204] Inhibitors decrease or prevent probe-target interactions.Inhibitors can be any substance, e.g., moiety, molecule, compound orparticle. Preferably the inhibitor competes with a known bindinginteraction between target and probe. Inhibitors are a form ofmodulator. Inhibitors are also referred to herein as blocking agents orblockers.

[0205] In a specific embodiment, compounds that are potential inhibitorsof an agonist to a receptor are screened by testing for removal of aconformational change induced by the agonist when the receptor andagonist are also in the presence of an inhibitor candidate (the agonistcan be a natural molecule, synthetic, etc.).

[0206] Decorators

[0207] A decorator refers to a nonlinear-active substance (e.g.,molecule or particle) which can be bound to targets, probes ortarget-probe complexes, and allow detection and discrimination amongthem. Ideally, a decorator should not appreciably alter or participatein the target-probe reaction itself. A decorator is distinguished from anonlinear-active label, such as a SHG-active label,in that it possessesa specific binding affinity for the target, probe, or the target-probecomplex, while an SHG-label can be attached to, for example a biologicalcomponent, via specific chemical bonds or non-specific (e.g.,electrostatic) means. A decorator can be used to detect probe-targetcomplexes by its specific binding affinity (in some art, ‘molecularrecognition’) to the targets, probes or the target-probe complexes andto thereby discriminate among targets, probes and target-probe complexesin a surface-selective nonlinear optical technique. For example, adecorator which has a stronger affinity (larger binding constant) todouble-stranded DNA than single-stranded DNA can be used to detecthybridization with surface-attached probes since the amount of orienteddecorator will increase as hybridization proceeds betweensingle-stranded targets and probes. In a specific embodiment, decoratorsare constructs of a nonlinear-active species and another species, wherethe other species is a biological component (protein, antibody, etc.)and where the construct has a differential binding affinity amongprobes, targets and probe-target complexes to allow discrimination amongthem using a surface-selective nonlinear optical technique. Decoratorscan also be a non-natural molecules (e.g., synthetic chemical molecule,drug, etc.) with a nonlinear activity and which binds specifically(recognizes) targets, probes or target-probe complexes, and has adifferential binding affinity among the three to allow fordiscrimination among them using a surface-selective nonlinear opticaltechnique. In a specific embodiment, the decorator is dissolved orsuspended in a solution or aqueous phase containing the targetcomponent.

[0208] Exemplary decorator molecules or particles include, but are notlimited to, a biological component, a nucleic acid, protein, smallmolecule, biological cell, virus, liposome, receptor, agonist,antagonist, inhibitor, hormone, antibody, antigen, peptide, receptor,drug, enzyme, ligand, nucleoside, polynucleoside, carbohydrate, cDNA,hormone, allergen, cDNA, hapten, oligonucleotide, biotin, streptavidin,polynucleotide, oligosaccharide, peptide nucleic acid (PNA), or nucleicacid analog. In other non-limiting embodiments, decorators comprise amoiety in the family of, or that is, psoralen, ethidium bromide, methanephosphonate, phosphoramidate, propidium iodide, acridine,9-aminoacridine, acridine orange, chloroquine, pyrine, echinomycin,4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), succinimidylacridine-9-carboxylate, chloroquine, pyrine, echinomycin,4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), single-strandbinding protein (SSB), tripyrrole peptides, flavopiridol, or pyronin Y.

[0209] Indicators

[0210] The following section describes indicators in detail. Indicatorsinclude nonlinear-active molecules or particles whose nonlinear opticalproperties or orientation near a surface or interface is modulated asthe electric charge polarization, charge density or potential of thesurface is modulated. In one aspect of the invention, the charge orpotential of an interface is modulated by the binding of a target toprobes immobilized on the surface. In another aspect, the surfaceelectric potential of a cell is changed by a change in the ion channelproperties—an opening, closing, increase or decrease in ionicpermeability in response to target (ligand) binding, for instance. Inanother aspect of the invention, an indicator serves as a marker forimaging purposes, e.g., to image cells or tissues. An indicatorpreferably does not appreciably alter or participate in the target-probereaction itself. The indicator can be dissolved or suspended in theliquid, medium, solution or aqueous phase containing the targetcomponent. An indicator preferably does not translocate into the lipidbilayer of vesicles or cells. An indicator preferably possesses freedomof movement to respond to changes in surface electric charge density orpotential.

[0211] Measuring the nonlinear optical response of a glass-solvent orglass-water interface, in the presence of dissolved or suspendedindicators in the water or solvent, is one means of assaying whether acandidate molecule would function as an indicator. Since glass carries anet negative charge, a candidate molecule can function as an indicatorif the intensity of the nonlinear optical radiation generated at theinterface in the presence of the molecule is greater than the backgroundsignal in its absence. Another means of assaying for a candidatemolecule's ability as an indicator is by measuring the intensity ofnonlinear optical radiation generated by a semiconductor-liquidinterface as a function of applied voltage (and hence surface electriccharge density) between the semiconductor and the bulk of the liquid.Yet another means is to measure the hyper-rayleigh scattering (HRS) froma solution or suspension of the indicator candidates, since if HRS isgenerated and the candidate itself is charged or dipolar, it shouldserve as an indicator.

[0212] Oxazole dye 4-[5-methoxyphenyl)-2-oxazolyl]pyridiniummethanesulfonate (also known as 4PyMPO-MeMs) is an indicator that can beused, that is strongly second harmonic-active and chemically stable atneutral pH (Salafsky and Eisenthal, Chemical Physics Letters 2000, 319,435-439). Furthermore, the Stokes shift of the fluorescence whichresults from two-photon absorption is large so that the second harmonicbeam can readily be separated from the fluorescence. Other dyes in thisfamily have similar properties (J. H. Hall et al., “Syntheses andPhotophysical Properties of Some 5(2)-Aryl-2(5)-(4-pyridyl)oxazoles andRelated Oxadiazoles and Furans”, J. Heterocyclic Chem. 29, 1245 (1992)).These and other molecules, or assemblies of the molecules, can be usedas indicators in the present invention. Such molecules include, but arenot limited to:

[0213] 5-(4-methoxphenyl)-2-(4-methoxyphenyl)-2-(4-pyridyl)oxazole

[0214] 2-(4-methoxyphenyl)-5-(4-pyridyl)oxazole

[0215] 2-(4-methoxyphenyl)-5-(4-pyridyl)oxadiazole

[0216] 2-(4-methoxyphenyl)-5-(4-pyridyl)furan

[0217] 2-(4-pyridyl)-4,5-dihydronapthol[1,2-d]-1,3-oxazole

[0218] 5-Aryl-2-(4-pyridyl)-4-R-oxazole where R is a hydrogen atom,methyl group, ethyl group or other akyl group.

[0219] 2-(4-pyridyl)cycloalkano[d]oxazole

[0220] 2-(4-pyridyl)phenanthreno [9,10-d]-1,3-oxazole

[0221] 6-Methoxy-4,4-dimethyl-2-(4-pyridyl)indeno[2,1-d]oxazole

[0222] 4,5-Dihydro-7-methoxy-2-(4-pyridyl)napthol[1,2-d]-1,3-oxazole

[0223] Other molecules or molecules of the following families which canbe used as indicators, include, but are not limited to:

[0224] Merocyanines

[0225] Stilbenes

[0226] Indodicarbocyanines

[0227] Hemicyanines

[0228] Stilbazims

[0229] Azo dyes

[0230] Cyanines

[0231] Stryryl-based dyes

[0232] Methylene blue

[0233] Diaminobenzene compounds

[0234] Polyenes

[0235] Diazostilbenes

[0236] Tricyanovinyl aniline

[0237] Tricyanovinyl azo

[0238] Melamines

[0239] Phenothiazine-stilbazole

[0240] Polyimide

[0241] Sulphonyl-substituted azobenzenes

[0242] Indandione-1,3-pyidinium betaine

[0243] Fluorescein

[0244] Benzooxazole

[0245] Perylene

[0246] Polymethacrylates

[0247] Oxonol

[0248] Derivatized Particle Indicators

[0249] A solid microparticle or a nanoparticle of size nanometers tomicrons in scale including, but not limited to, a sphere (latex,polystyrene, silica, etc.) or a strip, offers a surface area which canbe derivatized with a nonlinear-active moiety via chemical orelectrostatic means so that the entire object has a much higherhyperpolarizability than can be obtained otherwise. For instance,nonlinear-active dyes can be ordered on silica bead surfaces viaelectrostatic interactions (dye is positively charged, silica surface isnegatively charged) and the entire bead, if derivatized withtarget-reactive linkers, can then function as an indicator. If thenonlinear-active moieties can be aligned on the solid surface so thatphase interference between moieties is small, the overallhyperpolarizability will scale nonlinearly (eg., quadratically) in theirnumber. The solid particle can vary in shape and its size can range fromnanometers to microns in scale. Examples of the particles to be usedinclude, but are not limited to, polystyrene beads and silica beads,both readily commercially available.

[0250] a. Covalent Attachment

[0251] The solid particles to be used as indicators can besurface-derivatized using a variety of chemistries available in theprior art. Nonlinear-active moieties can be covalently coupled either tothe solid particles or to a derivatized layer.

[0252] For instance, polystyrene beads can be derivatized with dextran,lactose or amines (the latter case for example, via chloromethyl groupswith ethylenediamine). Silica can be derivatized using organofunctionalsilanes, for example using trichlorosilanes or other functional silanes(such as methoxy, amine, or other functional groups), to producesurfaces with a variety of chemical functionalities. The surfaces of thederivatized beads can then be reacted with a nonlinear-active moiety viaappropriate chemistry to produce the indicator.

[0253] b. Electrostatic Attachment

[0254] Nonlinear-active moieties can also be electrostatically bound toa micron- or nanometer-sized particle surface to produce indicators withlarge hyperpolarizabilities. A charged nonlinear-active moiety, anorganic dye for example, can be oriented at a counter-chargedmicroparticle surface, thus allowing for a net hyperpolarizability ofthe object when using an appropriate geometry. An example of anappropriate geometry is a microparticle sphere where the diameter isapproximately the wavelength of the fundamental light, i.e. from tens ofnanometers to microns so that destructive phase interference betweennonlinear-active moieties on opposing faces of the sphere is minimized.The hyperpolarizability of each dye at the spheres's surface, whenintegrated across the entire surface of the sphere of ˜wavelength oflight size, is large and positive. By way of example, but notlimitation, the following procedure can be used. Silica beads (˜200 nm,roughly spherical) are reacted with a low concentration of3-aminopropyltrimethoxysilane or 3-aminooctyltrimethoxysilane so thatonly ˜5-10% of the surface silanols become covalently coupled to thesilane agent. These amine groups are then reacted with theamine-reactive homobifunctional crosslinker disuccinimidyl glutarate(DSG, Pierce Chemical) to create amine-reactive linkers on ˜5-10% of thebead surface. The beads are then incubated with4-[5-methoxyphenyl)-2-oxazolyl]pyridinium methanesulfonate (also knownas 4PyMPO-MeMs), a positively charged dye which binds electrostaticallyto the charged silanols on the surface and orients to some degree. Theexcess dye is removed from the beads by centrifugation. Theelectrostatic adsorption can be sufficiently high in some cases toimmobilize the charged dye, even in the absence of a bulk concentrationof it.

[0255] Many nonlinear-active species are known in the art that can beused and include, but are not limited to, the following and theirderivatives:

[0256] Oxazole or Oxadizole Molecules

[0257] 5-aryl-2-(4-pyridyl)oxazole

[0258] 2-aryl-5-(4-pyridyl)oxazole

[0259] 2-(4-pyridyl)cycloalkano[d]oxazoles

[0260] Merocyanines

[0261] Stilbenes

[0262] Indodicarbocyanines

[0263] Hemicyanines

[0264] Stilbazims

[0265] Azo dyes

[0266] Cyanines

[0267] Stryryl-based dyes

[0268] Methylene blue

[0269] Diaminobenzene compounds

[0270] Polyenes

[0271] Diazostilbenes

[0272] Tricyanovinyl aniline

[0273] Tricyanovinyl azo

[0274] Melamines

[0275] Phenothiazine-stilbazole

[0276] Polyimides

[0277] Sulphonyl-substituted azobenzenes

[0278] Indandione-1,3-pyidinium betaine

[0279] Fluoresceins

[0280] Benzooxazoles

[0281] Perylenes

[0282] Polymethacrylates

[0283] Oxonols

[0284] Thiophenes

[0285] Bithiophenes

[0286] In evaluating whether a species may be nonlinear-active, thefollowing characteristics can indicate the potential for nonlinearactivity: a large difference dipole moment (difference in dipole momentbetween the ground and excited states of the molecule), a large Stokesshift in fluorescence, an aromatic or conjugated bonding character. Infurther evaluating such a species, an experimenter can use a simpletechnique known to those skilled in the art to confirm the nonlinearactivity using, for example, detection of SHG from an air-waterinterface or from EFISH in the absence and presence of the species inquestion in a medium. Once a suitable nonlinear-active species has beenselected for the experiment at hand, the species can be conjugated, ifdesired, to a species with specificity to a biological target to producea targeting construct used in the surface-selective nonlinear opticaldetection or imaging technique.

[0287] Enhancers

[0288] An enhancer as used herein refers to a substance (e.g., moiety,molecule or particle) which can enhance (increase) the cross-section ofa nonlinear-active substance (e.g., moiety, molecule or particle) whenplaced near to it (e.g., increase the intensity of second harmonicradiation generated). Examples of the enhancement effect referred to inthe art include ‘resonance enhancement’ and ‘surface enhancement’.Enhancement of the nonlinear-active cross-section moiety, molecule orparticle can occur via a resonance with an electronic transition orplasmon resonance of the enhancer. The addition of an enhancer onto, ornear to, a molecule or surface, can result in the enhancer coupling tothe molecule or surface, e.g., covalently, electrostatically,non-covalently, etc., or the enhancer is not coupled directly to themolecule or surface, but rather is near-to the molecule or surface,e.g., enhancer adsorption on to a cell surface, causing the enhancer toincrease the nonlinear response of the label on the probes.

[0289] The following (and references therein) describe the production,design and use of resonance-enhancing particles, such as metalnanoparticles, for nonlinear optical processes (SHG, Surface-enhancedresonance Raman, etc.): S. Nie and S. Emory, Science, 1997, 75, 1102; P.V. Kamat, M. Flumiani, G. V. Hartland, J. Phys. Chem. B, 1998, 102,3123; H. Ditlbacher et al., Appl. Phys. B, 2001, DOI: 10,1007/s003400100700; C. Sonnichsen et al., Appl. Phys. Lett., 2000, 77,2949; B. Lamprecht et al., Appl. Phys. B, 1997, 64, 269; J. P. Novak, J.Am. Chem. Soc., 2000, 122, 12029; S. R. Emory, W. E. Haskins, S.Nie, J.Am. Chem. Soc. 1998, 120, 8009; W. P. McConnell et al., J. Phys. Chem. B2000, 104, 8925; F. W. Vance, B. I. Lemon, J. T. Hupp, J. Phys. Chem. B1998, 102, 10091; P. Galletto et al., J. Phys. Chem. B 1999, 103, 8706;S. R. Emory, S. Nie, J. Phys. Chem. B 1998, 102, 493

[0290] The presence of the resonance-enhancing or surface-enhancingspecies serves to increase the nonlinear-active cross-section ofsamples. Examples of resonance-enhancing species in the art are thefollowing: metal or metallic (e.g., gold and silver) nanoparticles orcolloidal particles, metal-coated particles (e.g., silver-coated latexnanospheres), aggregates or clusters of any of the aforementioned,rationally-designed clusters, chains or aggregates of the aforementioned(e.g., for symmetry-breaking: non-centrosymmetric aggregates, particlesor clusters), etc.

[0291] In some instances, experimentation may be required to determinethe optimal labeling strategy and/or use of enhancers or decorators fora given measurement. For instance, the coupling chemistry, reactionconditions, etc. may be adjusted empirically to determine the optimallabeling strategy. Candidates for an enhancer can be tested for theireffect on a nonlinear-active species by measuring, for example, the SHGintensity of a nonlinear-active species in the absence and presence ofthe candidate enhancer (the enhancer can be attached to thenonlinear-active label, through a linker if necessary; or the enhancercan be brought into proximity to the label, e.g. by virtue of theprobe-target reaction). The enhancer's effect on a nonlinear-activelabel can be made at an interface (e.g., air-water or solid-liquid) orin bulk phase under the application of an electric field (EFISH).

[0292] Molecular Beacon Analogues

[0293] The nonlinear activity of a system can also be manipulatedthrough the introduction of nonlinear analogues to molecular beacons,that is, molecular beacon probes that have been modified to incorporatea nonlinear-active label (or modulator thereof) instead of fluorophoresand quenchers. These nonlinear optical analogues of molecular beaconsare referred to herein as molecular beacon analogues (MB analogues orMBA). The MB analogues to be used in the invention can be synthesizedaccording to procedures known to one of ordinary skill in the art.

[0294] In specific embodiments, the MB analogue probes can be usedaccording to the present invention as hybridization probes that canreport the presence of complementary nucleic acid targets without havingto separate probe-target hybrids from excess probes in hybridizationassays and without the need to label the targets. Target labeling is notonly time-consuming, but it can change the levels of targets originallypresent in a sample. MB analogue probes can also be used for thedetection of RNAs within living cells, for monitoring the synthesis ofspecific nucleic acids in sealed reaction vessels, and for theconstruction of self-reporting oligonucleotide arrays. They can be usedto perform homogeneous one-tube assays for the identification ofsingle-nucleotide variations in DNA and for the detection of pathogensor cells immobilized to surfaces for interfacial detection.

[0295]FIGS. 20A and 20B illustrate an embodiment of a MB analogue probe.A species with a hyperpolarizability capable of generating nonlinearoptical radiation in response to illumination with one or morefundamental beams is attached to one end of a probe or at one location(e.g., a dye molecule, see FIG. 20B). At the other end of the probe orin another location in proximity to the nonlinear-active label, aspecies capable of increasing or decreasing the nonlinear opticalradiation generated by a nonlinear-active label when the two species arein proximity is attached (e.g., a 10 nm gold particle, see FIG. 20B).The attachment can be via a covalent bond or through some other wellknown means in the art. In a specific embodiment, the nonlinear-activelabel is an organic dye with a hyperpolarizability such as the wellknown oxazole dyes and their derivatives. The oxazole dyes and theirderivatives are commercially available from Molecular Probes (Eugene,Oreg.) for attachment to a variety of probes including nucleic acidsusing amine and sulfhydryl chemistries. A species such as a goldnanoparticle, well known for its ability to enhance the nonlinearoptical radiation generated by a nonlinear-active species to which it isin proximity, can be used as the species capable of modulating thenonlinear optical radiation generated by the label. In specificembodiments, the MB analogue probes can be immobilized to a solidsurface such as a planar glass surface or to the surface ofmicrospheres, or the MB analogue probes can be used in homogeneoussolution and detected using an applied electric field in an EFISHmeasurement. If the MBAs are immobilized to a surface, thenonlinear-active species becomes at least partially oriented at thesurface and satisfies the non-centrosymmetric condition required forsurface-selective nonlinear optical techniques.

[0296] The Au nanoparticle can enhance the intensity of nonlinearoptical radiation, such as second harmonic generation scattered by theoxazole dye by several orders of magnitude when the nanoparticle andoxazole dye are in proximity to each other. Upon hybridization of theprobe to a complementary target, the intensity of the nonlinear opticalradiation decreases and this decrease can be quantitatively related tothe amount of probe-target hybridization. The sensitivity of thetechnique is determined by, among other factors, the backgroundnonlinear optical signal before hybridization occurs. The sequences ofvarious targets used for testing the degree of hybridization to theprobe in FIG. 20B are Target 1-4 in FIG. 20 (SEQ ID NOs: 1-4respectively).

[0297] The present invention can be used for detection of singlenucleotide polymorphisms (SNPs) in target samples because the MBA probesare highly selective in their binding of targets and a one base pairdifference in sequence between probes and targets will yield a muchreduced hybridization affinity compared with a target that is perfectlycomplementary. The MBA probe can act also as a label.

5.1 ASSAYING PROBE-TARGET INTERACTIONS

[0298] Throughout the description of the present invention testmolecules are also referred to as “targets,” and candidate bindingpartners are also referred to herein as “probes.” The following areexamples of the types of probe-target interactions that can be assayedaccording to the present invention:

[0299] i) Probe-target binding that results in a conformational changein the probe, target or both.

[0300] ii) Probe-target binding that results in a change in the dipolemoment of a nonlinear-active species, said species being the probe,target or both, as a result of probe-target binding.

[0301] Probe-target binding can also result in a combination of bothconformational and dipole moment changes.

[0302] Furthermore, detection of the probe-target interactions can occurat an interface, in bulk phase (homogeneous phase), or in regions thatcomprise a combination of interface and bulk.

[0303] The present invention can be applied to an ensemble of moleculesor to a single molecule—i.e., ensemble reaction measurements or asingle-molecule reaction measurement.

[0304] In a preferred embodiment, the target is a G protein-coupledreceptor (GPCR). GPCRs are one class of proteins that undergo aconformational change when activated by a ligand and are thus amenableto study using the present invention. In this case, if the GPCR is notintrinsically nonlinear-active, the protein is labeled using anonlinear-active label, and the conformational change is detected orqueried for via a change in the orientation of the nonlinear-activelabel. The GPCRs can be attached to a surface and the conformationalchange that results when a ligand activates the receptor causes a changein the orientation of the label and thus a change in properties of thenonlinear optical beams (e.g., second harmonic generation) such asintensity, wavelength or polarization. A background signal can bemeasured before exposure of the sample to a ligand, if desired.

[0305] In some cases, binding of a component to a receptor will lead toa change in measured nonlinear optical properties even though thereceptor is not activated. For example, this can be due to aninteraction between the component and the receptor in the bound complexwhich alters the orientation of a label attached to the receptor. Acontrol can be performed, if desired, to assign measured changes innonlinear optical properties to binding or activation of components to agiven receptor. For example, a component which is known to bind to agiven receptor but not to produce a conformational change can be used asa control of the label reaction; if any measured change in labelorientation is due only to receptor activation, the position of thelabel can be changed by changing the conjugation chemistry of the labeland/or genetically modifying the receptor to introduce new labelingsites.

[0306] For example, receptors can be labeled with a nonlinear-activelabel that possesses, on average, an orientation (or orientation ofhyperpolarizability) of some angle θ with respect of the surface plane(e.g., host cell membrane). Upon binding and/or activation to someligand, the average label angle can shift to angle β with respect to thesurface plane. Even small shifts in angle as a result of receptorbinding or activation can cause substantial changes in a property of themeasured nonlinear optical light (e.g., intensity of the nonlinearlight). For example, if the intensity of the nonlinear optical lightgenerated is proportional to the component of the hyperpolarizabilitythat is normal to the membrane plane, then the percentage change ofintensity upon a shift is: [cos(θ)/cos(β)]² with the nonlinear intensitydependent quadratically on the normal component of thehyperpolarizability. For example, assuming a delta function inhyperpolarizability orientation, if θ→β is a change of 25 degrees to 30degrees, the nonlinear optical intensity will decrease about 9%.

[0307] In some instances, some experimentation may be required todetermine the optimal labeling strategy and/or use of enhancers ordecorators for a given measurement. For instance, the couplingchemistry, reaction conditions, etc. may need to be adjusted empiricallyto determine the optimal labeling strategy.

[0308] Alternatively, the target molecules can be solubilized and, usingtheir intrinsic dipole moments, poled by an electric field in solutionphase; if the target possesses a hyperpolarizability (i.e., isnonlinear-active, either intrinsically or made so by attachment of anonlinear-active label), a nonlinear optical signal will be generatedvia EFISH technique, and this signals serves as the background. Uponaddition of a ligand (i.e., a probe) that activates the target molecule,a conformational change will occur. If this conformational change doesnot result in an appreciable dipole moment change in the targetmolecule, the number and net orientation of the target molecules willnot change appreciably. If, however, this conformational change ispassed to the nonlinear-active moiety, its overall orientation willchange, resulting in a change in measured nonlinear optical properties.If the binding of a ligand to the target molecules results in anappreciable dipole moment change of the nonlinear-active targetmolecule, the number of aligned target molecules in the applied fieldwill change, resulting in change in measured nonlinear opticalproperties. The number of aligned molecules in an applied electric fieldin solution is dependent on the dipole moment of the molecules. Anequation used to model the dependence is the Langevin equation: N=N₀exp(−μE/kT) where N is the number of aligned species, μ is their dipolemoment, E is the electric field magnitude parallel to the dipole moment,N₀ the number of molecules exposed to the field, k is the Boltzmannconstant and T is the temperature in Kelvin. Changes in dipole moment asa result of probe-target interactions can lead to large changes in thenumber of aligned molecules and thus large changes in the intensity ofnonlinear optical light generated by the molecules. For example, a probewith a nonlinear-active label and a given dipole moment binds to atarget; the resulting probe-target complex with a larger or smallerdipole moment leads to a change in intensity of the generated nonlinearlight. Measured changes as a result of binding can also be used tocalculate binding conformations between molecules if the position andamount of charge is well known on each molecule (e.g., as is often thecase with proteins, whose crystal structure is known).

[0309] The above illustrations are exemplary of any probe-targetinteraction that results in a change in dipole moment or conformationalchange. Ion channel proteins are examples of another important class ofproteins that undergo conformational change in response to activationand are also amenable to study using the present invention.

[0310] Surface-selective nonlinear optical techniques are also coherenttechniques, meaning that the fundamental and nonlinear beams havewell-defined phase relationships, and the wavefronts of a nonlinear beamin a macroscopic sample (within the coherence length) are in phase.These properties offer a number of advantages useful for surface orhigh-throughput studies in which, for example, either a single surfaceor a microarray surface is examined. An apparatus using nonlinearoptical suface-selective-based detection, such as with second harmonicgeneration, requires minimal collection optics since generation of thenonlinear light only occurs at the interface and thus, in principle,allows extremely high depth discrimination and fast scanning.

[0311] Probe-target interactions (e.g., a binding reaction, aconformational change, etc.) can be correlated with the presentinvention to the following measurable information, for example:

[0312] i) the intensity of the nonlinear or fundamental light.

[0313] ii) the wavelength or spectrum of the nonlinear or fundamentallight.

[0314] iii) position of incidence of the fundamental light on thesurface or substrate (e.g., for imaging).

[0315] iv) the polarization of the nonlinear light

[0316] v) the time-course of i), ii), iii) or iv).

[0317] vi) one or more combinations of i), ii), iii), iv) and v).

[0318] The advantages of the present invention are enumerated asfollows:

[0319] i) Sensitive and direct dependence on the orientation and/ordipole moment of the nonlinear-active species in a sample, useful fordetection of conformational changes in probes and binding that resultsin an appreciable change in the dipole moment of the nonlinear-activespecies (i.e., probe, target or both).

[0320] ii) Higher signal to noise (lower background) thanfluorescence-based detection since surface-selective nonlinear opticallight is generated only at surfaces that create a non-centrosymmetricsituation, or in homogeneous phase under application of an electricfield to induce the non-centrosymmetry. surface-selective nonlinearoptical light detection of a surface has a very narrow ‘depth of field’.Sources of fluorescence in fluorescence-based detection schemes includethat from materials in the field of view but not in the focal plane,autofluorescence, and contamination of the emitted fluorescence withstray excitation light; these are not sources of background nonlinearoptical radiation.

[0321] iii) The nonlinear optical technique is useful when the presenceof a liquid solution is required for the measurement, i.e. where thebinding process can be obviated or disturbed by a wash-away step. Thisaspect of the invention can be useful for equilibrium measurements (freeenergy, binding constants, etc.), which require the presence of bulkspecies or kinetics measurements with measurements made over a period oftime.

[0322] iv) Lower photobleaching and heating effects than those thatoccur in fluorescence—the two-photon absorption cross-section is muchlower than the one-photon cross-section in a molecule and the nonlinearoptical technique involves scattering, not absorption.

[0323] v) A minimum of collection optics is needed and higher signal tonoise is expected since the fundamental and nonlinear beams (e.g.,second harmonic) have well-defined incoming and outgoing directions withrespect to the interface. This is advantageous compared tofluorescence-based detection in which the fluorescence is emittedisotropically and there may be a large auto-fluorescence background outof the plane of interest (e.g., the interface containing the probes).

[0324] vi) Ease of use with beads, biological cells, liposomes or otherparticles whose non-planar surface makes an interface with thesupporting medium, solution, etc.

[0325] vii) Convenience of discriminating between binding of targets toprobes from actual activation of probes (e.g., a receptor) by a target.

[0326] viii) The binding process between probes and targets can beperformed in the presence of one or more small molecules, drugs,blocking agents, or other components which affect properties of theprobe-target binding process, e.g. equilibrium constants, kinetics ofbinding, etc.

[0327] In an embodiment, using surface arrays, arrays can be constructedaccording a plurality of methods found in the art. For DNA microarrays,most are prepared with one of three non-standard approaches (S. C.Case-Green et al., Curr. Opin. Chem. Biol. 2 (1998), 404): Affymetrix,Inc. probe arrays are prepared using patterned, light-directedcombinatorial chemical synthesis (S. A. Fodor, Science 277 (1997), 393);spotted arrays can be made according to D. H. Duggan et al., NatureGenet. 21 (Suppl.) (1999), 10; M. Schena et al., Science 270 (1995),467; P. O. Brown and D. Botstein, Nature Genet. 21 (Suppl.) (1999), 33;and L. McAllister et al., Am. J. Hum. Genet. 61 (Suppl.) (1997), 1387;ink-jet techniques can also be used to synthesize oligonucleotides baseby base through sequential solution-based reactions on an appropriatesubstrate (A. P. Blanchard et al., Biosens. And Bioelectron. 11 (1996)687-relevant portions of all of which references are incorporated byreference herein).

[0328] For example, nucleic acid, oligo- or nucleotide arrays can beconstructed according to U.S. Pat. No. 6,110,426, U.S. Pat. No.5,143,8546,110,426—relevant portions of which are incorporated byreference herein, U.S. Pat. No. 5,143,854—relevant portions of which areincorporated by reference herein or Fodor et al., “Light-directedSpatially-addressable Parallel Chemical Synthesis,” Science, 1991, 251,767-773. Soluble protein arrays can be constructed according to R.Ekins, F. W. Chu, Trends in Biotechnology, 1999, 17, 217, relevantportions of which are incorporated by reference herein. Membraneproteins arrays can be constucted by micropatterning of fluid lipidmembranes according, for example, to the method of Groves, J. T., Ulman,N., Boxer, S. G., “Micropatterning fluid bilayers on solid supports”,Science, 1997, 275, 651-3 (relevant portions of which are incorporatedby reference herein). The array substrate can be composed of glass,silicon, indium tin oxide, or any other substrate known in the art. Thesurface array under study can contain physical barriers between elementsso that the elements (and their biomolecules) can remain in isolationfrom each other during a chemical reaction step. The array locations canconsist of different probes, the same probes everywhere, or somecombination thereof. The array can also be constructed on the undersideof a prism allowing. for total internal reflection of the beam andevanescent generation of the nonlinear light. Or an array substrate canbe brought into contact with a prism with the same result.

[0329] An electrophoretic system can also be used in conjunction withthe surface array, for example to deliver reagents or biologicalcomponents to one or a plurality of locations using flow channels ormicrocapillaries. The sample can include an array of microcapillarychannels, each distinct from the other and each allowing a target-probereaction to occur; the imaging technique would then consist of arrayelements, each one a microcapillary channel or reaction chamber intowhich the channel feeds or drains.

[0330] The polarization of the fundamental and nonlinear beams can beselected with polarizing optics elements. By analyzing the intensity ofthe nonlinear beam as a function of fundamental and nonlinearpolarization, more information (e.g., higher signal to noise) about theprobe-target complexes can be obtained. Furthermore, by selecting andanalyzing the polarization of the fundamental or nonlinear opticalradiation, background radiation can be reduced or signal intensityenhanced.

[0331] Detection can be accomplished with the use of multiple internalreflection plates (N. J. Harrick, “Internal Reflection Spectroscopy”,John Wiley & Sons, Inc., New York, 1979—relevant portions of which areincorporated by reference herein) allowing the fundamental beam to makemultiple contacts with the array surface, thus increasing the intensityof the generated nonlinear light. Another alternative is to construct anoptical cavity with the array surface on one side and a lossy coupler atone end to permit the output coupling of the nonlinear light, creatingan optical microcavity which would allow the buildup of very highintensities under resonance and thus increase the amount of nonlinearlight generated.

[0332] Polynucleotide arrays can be used as probes. Whereoligonucleotides are targets or probes, preferably a nonlinear-activelabel is attached to the 5′ or 3′ termini. There are many linkingmoieties and methodologies for attaching molecules which can benonlinear-active labels to the 5′ or 3′ termini of oligonucleotides, asexemplified by the following references: Eckstein, editor,Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford,1991); Zuckerman etal., Nucleic Acids Research, 15: 5305-5321 (1987) (3′thiol group on oligonucleotide); Sharma et al., Nucleic Acids Research,19: 3019 (1991) (3′ sulfhydryl); Giusti et al., PCR Methods andApplications, 2: 223-227 (1993) and Fung et al., U.S. Pat. No. 4,757,141(5′ phosphoamino group via Aminolink.TM. II available from AppliedBiosystems, Foster City, Calif.) Stabinsky, U.S. Pat. No. 4.739,044 (3′aminoalkylphosphoryl group); Agrawal et al., Tetrahedron Letters, 31:1543-1546 (1990) (attachment via phosphoramidate linkages); Sproat etal., Nucleic Acids Research, 15: 4837 (1987) (5′ mercapto group); Nelsonet al., Nucleic Acids Research, 17: 7187-7194 (1989) (3′ amino group);and the like, relevant portions of which are incorporated by referenceherein.

[0333] Preferably, commercially available linking moieties are employedthat can be used to a label to an oligonucleotide during synthesis,e.g., available from Clontech Laboratories (Palo Alto, Calif.). In aspecific embodiment, rhodamine and fluorescein dyes can be convenientlyattached to the 5′ hydroxyl of an oligonucleotide at the conclusion ofsolid phase synthesis by way of dyes derivatized with a phosphoramiditemoiety, e.g., Woo et al., U.S. Pat. No. 5,231, 191; and Hobbs, Jr., U.S.Pat. No. 4,997,928, relevant portions of which are incorporated byreference herein.

[0334] Preferable, the oligonucleotides are present on arrays.

[0335] Protein arrays can be used to determine whether a given targetprotein binds to the immobilized probe protein on the surface; thesearrays can also be used to study small molecule binding to the probeproteins. Protein arrays can be prepared by the method of G. MacBeathand S. L. Schreiber, “Printing Proteins as Microarrays forHigh-Throughput Function Determination”, Science 2000, 289, 1760-1763,for example, to determine whether a given target protein binds to theimmobilized probe protein on the surface.

[0336] The surface on which the probes are formed may be composed from awide range of material, either biological, non-biological, organic,inorganic, or a combination of any of these, existing as particles,strands, precipitates, gels, sheets, tubing, spheres, containers,capillaries, pads, slices, films, plates, slides, etc. The surface mayhave any convenient shape, such as a disc, square, sphere, circle, etc.The surface is preferably flat but may take on a variety of alternativesurface configurations. For example, the surface may contain raised ordepressed regions on which a sample is located. The surface and itssurface preferably form a rigid support on which the sample can beformed. The surface and its surface are also chosen to provideappropriate light-absorbing characteristics. For instance, the surfacemay be a polymerized Langmuir Blodgett film, functionalized glass, Si,Ge, GaAs, GaP, Si_(x)Oy, Si_(x)N_(y), modified silicon, or any one of awide variety of gels or polymers such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinationsthereof. Other surface materials will be readily apparent to those ofskill in the art upon review of this disclosure. In a preferredembodiment the surface is flat glass or silica.

[0337] According to some embodiments, the surface of the substrate isetched using well known techniques to provide for desired surfacefeatures. For example, by way of the formation of trenches, v-grooves,mesa structures, or the like, the synthesis regions may be more closelyplaced within the focus point of impinging light. The surface may alsobe provided with reflective “mirror” structures for maximization ofemission collected therefrom.

[0338] The chemical identity of the probes (e.g., protein structure oroligonucleotide sequence) can vary from site to site across the solidsurface, or the same probe can uniformly cover the surface. Targets canbe of a single identity or a combination of targets with differentidentities.

[0339] In a specific embodiment, the kinetics of probe-target bindingreactions are measured as a function of target concentration. In thisembodiment, the time course of the intensity and/or spectrum of thenonlinear optical light are measured. The measured information isconverted into a time course of bound target concentration (e.g.,probe-target concentration in mM/s or μM/s). Drugs or modulators of theprobe-target binding equilibrium or kinetic rate of formation can beused so as to compare the effect of the added substance on theprobe-target reactions.

[0340] Various art not involving the use of a surface-selectivenonlinear optical technique contains relevant portions for the presentinvention and the following exemplary list and their references thereinis referenced herein: King et al.., U.S. Pat. No. 5,633,724 for thescanning and analysis of the scans; Fork et al., U.S. Pat. No. 6,121,983for the multiplexing of a laser to produce a laser array suitable forscanning; Foster, U.S. Pat. No. 5,485,277; Fodor et al., U.S. Pat. No.5,324,633 and Fodor et al., U.S. Pat. No. 6,124,102 for a substratecontaining an array of attached probes and for the analysis of scans todetermine kinetic and equilibrium properties of a binding reactionbetween probes and targets; Kain et al., U.S. Pat. No. 5,847,400 forlaser scanning of a substrate; King et al., U.S. Pat. No. 5,432,610 foran optical resonance cavity for power build-up; Walt et al., U.S. Pat.No. 5,320,814, Walt et al., U.S. Pat. No. 5,250,264, Walt et al., U.S.Pat. No. 5,298,741, Walt et al., U.S. Pat. No. 5,252,494, Walt et al.,U.S. Pat. No. 6,023,540, Walt et al., U.S. Pat. No. 5,814,524, Walt etal., U.S. Pat. No. 5,244,813 for fiber-optic-based apparatus; Fiekowskyet al., U.S. Pat. No. 6,095,555 for imaging and software-based analysisof images; Stem et al., U.S. Pat. No. 5,631,734 for data acquisition;Stimson et al., U.S. Pat. No. 6,134,002 for confocal imaging techniques;Sampas, U.S. Pat. No. 6,084,991 for CCD-based imaging techniques; Stemet al., U.S. Pat. No. 5,631,734 for photolithographical preparation ofprobes attached to surfaces; Shalon et al., U.S. Pat. No. 6,110,426 formethods and apparatus for creating attached probes on a surface;Slettnes, U.S. Pat. No. 6,040,586 for position-based scanningtechniques; Trulson et al, U.S. Pat. No. 6,025,601 for methods ofimaging probe-target binding on a surface.

[0341] Cells Attached to Surfaces and Microarrays of Cells

[0342] This section outlines some of the methods concerned withinterfacing biological cells with surfaces and fabricating arrays ofbiological cells on surfaces, which can be used in the assays of thepresent invention. Many methods have been described for making uniformmicro-patterned arrays of cells for other applications, using forexample photochemical resist-photolithograpy. (Mrksich and Whitesides,Ann. Rev. Biophys. Biomol. Struct. 25:55-78, 1996). According to thisphotoresist method, a glass plate is uniformly coated with a photoresistand a photo mask is placed over the photoresist coating to define the“array” or pattern desired. Upon exposure to light, the photoresist inthe unmasked areas is removed. The entire photolithographically definedsurface is uniformly coated with a hydrophobic substance such as anorganosilane that binds both to the areas of exposed glass and theareas-covered with the photoresist. The photoresist is then strippedfrom the glass surface, exposing an array of spots of exposed glass. Theglass plate then is washed with an organosilane having terminalhydrophilic groups or chemically reactable groups such as amino groups.The hydrophobic organosilane binds to the spots of exposed glass withthe resulting glass plate having an array of hydrophilic or reactablespots (located in the areas of the original photoresist) across ahydrophobic surface. The array of spots of hydrophilic groups provides asubstrate for non-specific and non-covalent binding of certain cells,including those of neuronal origin (Klienfeld et al., J. Neurosci.8:4098-4120, 1988). Reactive ion etching has been similarly used on thesurface of silicon wafers to produce surfaces patterned with twodifferent types of texture (Craighead et al., Appl. Phys. Lett. 37:653,1980; Craighead et al., J. Vac. Sci. Technol. 20:316, 1982; Suh et al.Proc. SPIE 382:199, 1983).

[0343] In another method based on specific yet non-covalentinteractions, photoresist stamping is used to produce a gold surfacecoated with protein adsorptive alkanethiol. (Singhvi et al., Science264:696-698, 1994). The bare gold surface is then coated withpolyethylene-terminated alkanethiols that resist protein adsorption.After exposure of the entire surface to laminin, a cell-binding proteinfound in the extracellular matrix, living hepatocytes attach uniformlyto, and grow upon, the laminin coated islands (Singhvi et al. 1994). Anelaboration involving strong, but non-covalent, metal chelation has beenused to coat gold surfaces with patterns of specific proteins (Sigal etal., Anal. Chem. 68:490-497, 1996). In this case, the gold surface ispatterned with alkanethiols terminated with nitriloacetic acid. Bareregions of gold are coated with tri(ethyleneglycol) to reduce proteinadsorption. After adding Ni²⁺, the specific adsorption of fivehistidine-tagged proteins is found to be kinetically stable.

[0344] More specific uniform cell-binding can be achieved by chemicallycrosslinking specific molecules, such as proteins, to reactable sites onthe patterned substrate (Aplin and Hughes, Analyt. Biochem. 113:144-148,1981). Another elaboration of substrate patterning optically creates anarray of reactable spots. A glass plate is washed with an organosilanethat chemisorbs to the glass to coat the glass. The organosilane coatingis irradiated by deep UV light through an optical mask that defines apattern of an array. The irradiation cleaves the Si—C bond to form areactive Si radical. Reaction with water causes the Si radicals to formpolar silanol groups. The polar silanol groups constitute spots on thearray and are further modified to couple other reactable molecules tothe spots, as disclosed in U.S. Pat. No. 5,324,591, incorporated byreference herein. For example, a silane containing a biologicallyfunctional group such as a free amino moiety can be reacted with thesilanol groups. The free amino groups can then be used as sites-ofcovalent attachment for biomolecules such as proteins, nucleic acids,carbohydrates, and lipids. Other methods for patterning the adhesion ofmammalian cells to surfaces using self-assembled monolayers on a surfaceinclude Lopez et al., “Convenient Methods for Patterning the Adhesion ofMammalian Cells to Surfaces Using Self-Assembled Monolayers ofAlkanethiolates on Gold,” J. Am. Chem. Soc., 1993, 115, 5877-5878,Georger et al., “Coplanar Patterns of Self-assembled Monolayers forSelective Cell-adhesion and Outgrowth,”Thin Solid Films 210 (1-2):716-719 Apr. 30, 1992, and Spargo et al., PNAS 91 (23): 11070-11074(1994).

[0345] The non-patterned covalent attachment of a lectin, known tointeract with the surface of cells, to a glass substrate throughreactive amino groups has been demonstrated (Aplin and Hughes, Analyt.Biochem. 113:144-148, 1981). The optical method of forming a uniformarray of cells on a support requires fewer steps and is faster than thephotoresist method, (i.e., only two steps), but it requires the use ofhigh intensity ultraviolet light from an expensive light source.

[0346] Cells can also be cultured or grown on surfaces that require noadditional derivatization. Surfaces of this type well known in the artinclude Becton-Dickinson Falcon plates and others.

[0347] In all of these methods the resulting array of cells orsurface-attached cell layer is uniform. In the photoresist method, cellsbind to the array of hydrophilic spots and/or specific moleculesattached to the spots, which, in turn, bind cells. Thus cells bind toall spots in the array in the same manner. In the optical method, cellsbind to the array of spots of free amino groups by adhesion. Methods forattaching a variety of cell types to the same substrate forsimultaneously binding against these cell types also exist and can beused.

[0348] Nucleic Acid Arrays

[0349] Nucleic acid arrays are useful in a number of biological andclinical studies in which one or more genes are analyzed in parallelusing the array. Genetic disease is often caused by genes that areinappropriately transcribed—either too much or too little—or which aremissing altogether. Such defects are especially common in cancers, whichcan occur when regulatory genes are deleted, inactivated, or becomeconstitutively active. Unlike some genetic diseases (e.g. cysticfibrosis) in which a single defective gene is always responsible,cancers that appear clinically similar can be genetically heterogeneous.For example, prostate cancer (prostatic adenocarcinoma) may be caused byseveral different, independent regulatory gene defects even in a singlepatient. In a group of prostate cancer patients, every one may have adifferent set of missing or damaged genes, with differing implicationsfor prognosis and treatment of the disease.

[0350] Comparative hybridization can serve two purposes in studyingcancer: it can pinpoint the transcription differences responsible forthe change from normal to cancerous cells, and it can distinguishdifferent patterns of abnormal transcription in heterogeneous cancers.Understanding the diverse basis of a cancer is crucial for inventingtherapies targeted to the different varieties of the disease, so thateach patient receives the most appropriate and effective treatment.

[0351] Cancers are common examples of genetically heterogeneousdiseases, but they are by no means the only ones. Diabetes, heartdisease, and multiple sclerosis are among the diseases for which geneticrisk factors are known to be heterogeneous.

[0352] Peptide-Nucleic Acids

[0353] In an alternative embodiment, peptide nucleic acids or oligomers,which are analogs of nucleic acids in which, for example, thepeptide-like backbone is replaced with an uncharged backbone, can beused with the present invention. PNAs are well known in the art.References below give extensive reviews of the use of these nucleic acidanalogs in a wide range of applications, including surface andarray-based hybridization wherein PNAs are attached to surfaces andallowed to bind with sequence-complementary DNAs or RNAs.

[0354] For instance, oligomers of PNA can be used as thesurface-attached probe components instead of DNA oligomers. A keyadvantage to using PNAs is that the hybridization reaction with DNAs orRNAs, for example, (containing charged phosphate groups) is only weaklydependent (eg., the melting temperature) on ionic strength because thereis much less charge repulsion as found with conventional DNA-DNA, etc.hybridization. Thus, one can use the surface-selective nonlinear opticaltechnique to follow a probe-target hybridization at any desired ionicstrength. The PNAs are commercially available (for instance via AppliedBiosystems, Foster City, Calif.) or other analogs of DNA can besynthesized and used.

[0355] The following references are broad reviews of the use of PNAs:

[0356] Nielsen, et al. “Peptide nucleic acids (PNA): Oligonucleotideanalogues with a polyamide backbone” Antisense Research and Applications(1992) 363-372

[0357] Nielsen, et al. “Peptide nucleic acids (PNAs): PotentialAntisense and Anti-gene Agents.” Anti-Cancer Drug Design 8 (1993) 53-63

[0358] Buchardt, et al. “Peptide nucleic acids and their potentialapplications in biotechnology” TIBTECH 11 (1993) 384-386

[0359] Nielsen, P. E., Egholm, M. and Buchardt, O. “Peptide Nucleic Acid(PNA). A DNA mimic with a peptide backbone” Bioconjugate Chemistry 5(1994) 3-7

[0360] Nielsen “Peptide nucleic acid (PNA): A lead for gene therapeuticdrugs” Antisense Therapeutics 4 (1996) 76-84

[0361] Nielsen, P. E. “DNA analogues with nonphosphodiester backbones”Annu.Rev.Biophys.Biomol.Struct. 24 (1995) 167-183

[0362] Hyrup, B. and Nielsen, P. E. “Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications” Bioorg. Med. . 4(1996) 5-23

[0363] Mesmaeker, A. D., Altman, K.-H., Waldner, A. and Wendeborn, S.“Backbone modifications in oligonucleotides and peptide nucleic acidsystems” Curr. Opin. Struct. Biol. 5 (1995) 343-355

[0364] Noble, et al. “Impact on Biophysical Parameters on the BiologicalAssessment of Peptide Nucleic Acids, Antisense Inhibitors of GeneExpression” Drug.Develop.Res. 34 (1995) 184-195

[0365] Dueholm, K. L. and Nielsen, P. E. “Chemistry, properties, andapplications of PNA (Peptide Nucleic Acid)” New J. Chem. 21 (1997) 19-31

[0366] Knudsen and Nielsen “Application of Peptide Nucleic Acid inCancer Therapy” Anti-Cancer Drug 8 (1997) 113-118

[0367] Nielsen, P. E. “Design of Sequence-Specific DNA-Binding Ligands”Chem. Eur. J. 3 (1997) 505-508

[0368] Corey “Peptide nucleic acids: expanding the scope of nucleic acidrecognition” TIBTECH 15 (1997) 224-229

[0369] Nielsen, P. E. and Ørum, H. “Peptide nucleic acid (PNA), a newmolecular tool.” In Molecular Biology: Current Innovations and FutureTrends, Part2. Horizon Scientific Press, (1995) 73-89

[0370] Nielsen, P. E. and Haaima, G. “Peptide nucleic acid (PNA). A DNAmimic with a pseudopeptide backbone” Chem. Soc. Rev. (1997) 73-78

[0371] Ørum, H., Kessler, C. and Koch, T. “Peptide Nucleic Acid” NucleicAcid Amplification Technologies: Application to Disease Diagnostics(1997) 29-48

[0372] Wittung, P., Nielsen, P. and Norden, B. “Recognition ofdouble-stranded DNA by peptide nucleic acid” Nucleosid. Nucleotid. 16(1997) 599-602

[0373] Weisz, K. “Polyamides as artificial regulators of geneexpression” Angew. Chem. Int. Ed. Eng 36 (1997) 2592-2594

[0374] Nielsen, P. E. “Structural and Biological Properties of PeptideNucleic Acid (Pna)” Pure & Applied Chemistry 70 (1998) 105-110

[0375] Nielsen, P. E. “Sequence-specific recognition of double-strandedDNA by peptide nucleic acids” Advances in DNA Sequence-Specific Agents 3(1998) 267-278

[0376] Nielsen “Antisense Properties of Peptide Nucleic Acid” Handbookof Experimental Pharmacology 131 (1998) 545-560

[0377] Nielsen “Peptide Nucleic Acids” Science and Medicine (1998) 48-55

[0378] Uhlmann, E. “Peptide nucleic acids (PNA) and PNA-DNA chimeras:from high binding affinity towards biological function”Biol Chem 379(1998) 1045-52

[0379] Wang “DNA biosensors based on peptide nucleic acid (PNA)recognition layers. A review” Biosens Bioelectron 13 (1998) 757-62

[0380] Uhlmann, E., Peyman, A., Breipohl, G. and Will, D. W. “PNA:Synthetic polyamide nucleic acids with unusual binding properties”Angewandte Chemie-International Edition 37 (1998) 2797-2823

[0381] Nielsen, P. E. “Applications of peptide nucleic acids” Curr OpinBiotechnol 10 (1999) 71-75

[0382] Bakhtiar, R. “Peptide nucleic acids: deoxyribonucleic acid mimicswith a peptide backbone” Biochem. Educ. 26 (1998) 277-280

[0383] Lazurkin, Y. S. “Stability and specificity of triplexes formed bypeptide nucleic acid with DNA” Mol. Biol. 33 (1999) 79-83

[0384] Nielsen and Egholm “Peptide Nucleic Acids: Protocols andApplications” (1999) 266 pp.

[0385] Eldrup and Nielsen “Peptide nucleic acids: potential as antisenseand antigene drugs” Adv. Amino Acid Mimetics Peptidomimetics 2 (1999)221-245

[0386] Bentin, T. and Nielsen, P. E. “Triplexes involving PNA” TripleHelix Form. Oligonucleotides (1999) 245-255

[0387] Falkiewicz, B. “Peptide nucleic acids and their structuralmodifications” Acta Biochim. Pol. 46 (1999) 509-529.

[0388] The following references are descriptions of the use of PNAs inarray-based detection, including means for attaching the PNA probes tothe solid surface.

[0389] Hoffman, R., et al. “Low scale multiple array synthesis and DNAhybridization of peptide nucleic acids” Pept. Proc. Am. Pept. Symp.,15th (1999) 233-234

[0390] Matysiak, S., Hauser, N. C., Wurtz, S. and Hoheisel, J. D.“Improved solid supports and spacer/linker systems for the synthesis ofspatially addressable PNA-libraries” Nucleosides Nucleotides 18 (1999)1289-1291.

5.2 ADDITIONAL EMBODIMENTS

[0391] The drawings illustrate various specific embodiments of anapparatus and sample using second or higher harmonic, sum or differencefrequency generation.

[0392]FIG. 1 illustrates an embodiment wherein second harmonic light isgenerated by reflecting incident fundamental light from the surface.Light source 5 provides the fundamental light necessary to generatesecond harmonic light at the sample. Typically this will be a picosecondor femtosecond laser, either wavelength-tunable or not tunable, andcommercially available. Light at the fundamental frequency (ω) exits thelaser and its polarization is selected using, for example a half-waveplate 10 appropriate to the frequency and intensity of the light (eg.,available from Melles Griot, Oriel or Newport Corp.). The beam is thenfocused by lens 15 and passes through a pass filter 20 designed to passthe fundamental light but block the nonlinear light (eg., secondharmonic). This filter is used to prevent back-reflection of the secondharmonic beam into the laser cavity which can cause disturbances in thelasing properties. The beam is reflected from a mirror 25 and impingesat a specific location and with a specific angle θ on the surface. Themirror 25 can be scanned if required using a galvanometer-controlledmirror scanner, a rotating polygonal mirror scanner, a Bragg diffractor,acousto-optic deflector, or other means known in the art to allowcontrol of a mirror's position. The sample surface 30 can be mounted onan x-y translation stage 35 (computer controlled) to select a specificlocation on the surface for generation of the second harmonic beam. Thesurface can be glass, plastic, silicon or any other solid surface thatreflects the fundamental or second harmonic beams. The sample surfacecan be enclosed and the surface in contact with liquid. Furthermore, thesample 30 can be fed or drained by microcapillary or otherliquid-transporting channels (not shown), pumps or electrophoreticelements, and these devices can be computer-controlled. The fundamentaland the second harmonic outgoing beams (at specific angles with respectto the surface, i.e. θ₁—they are typically nearly colinear in direction)then reflected from the surface and the fundamental is filtered using apass-filter 45 for the second harmonic beam, leaving only the harmonicbeam (2ω). The second harmonic is reflected from mirror 40, itspolarization selected if necessary by polarizing optic 50, and isfocused using a lens 55 onto a detector 60. The lenses 15 and 55 canalso be any combination of lenses known in the art for focusing or beamshaping. If required, a mono chromator 60 can also be used to select aspecific wavelength within the spectral band of the second harmonicbeam. The detector can be a photomultiplier tube, a CCD array, or anyother detector device known in the art for high sensitivity. Forinstance, a photomultiplier tube operated in single-photon counting modecan be used. At the detector, the light generates a voltage proportionalto its intensity. Data is recorded for each location on the arraysurface as it is translated by the stage, scanned (or a combinationthereof) and an image is built up of the second harmonic intensitygenerated from each region on the surface.

[0393] Applicant envisions the use of sum or difference frequencies,where an apparatus set-up similar to FIG. 1 could be used, with thesingle light source 5 replaced by two light sources with two fundamentallight beams at frequencies ω₁, and ω₂. The sum or difference frequency(Ω) would then be Ω=ω₁±ω₂. In the case where the sample surfaces arearrays comprised of discrete elements, a single element or more than onein parallel can be addressed with the fundamental light. Furthermore,detection can be made on a single element or many in parallel dependingon the specific apparatus set-up.

[0394]FIG. 2 illustrates an embodiment in which total internalreflection (evanescent wave generation) is used to generate the secondharmonic light. Fundamental light (ω) is directed on to the surface of aprism element 70. The beam is refracted at position (a) and passesthrough the prism, through an index matching film 75 and impinges onsubstrate 80. Prism 70 and substrate 80 are made of opticallytransparent materials and are preferably of the same type. Prism 70 canbe a Dove prism or any other element which can support evanescent fields(eg., waveguides, fibers and thin metallic films). The refractive indexmatching film 75 can be an oil, but is preferably a compressible opticalpolymer such as those disclosed by Sjodin, “Optical interface means”,PCT publication WO 90/05317, 1990. The prism 75 and the substrate 80 canalso be a unitary, integral piece made of the same material (i.e,without the index matching film). An evanescent wave is generated at theinterface between 80 and the medium in sample compartment 85 accordingto the indices of refraction in 80 and 85 and the angle of incidence ofthe beam at their interface. The electric field amplitude decaysexponentially away from the substrate surface with a 1/e length rangingfrom nanometers to microns depending on several factors, including thesurface electric potential, the counterion density in the samplecompartment (if any). The sample compartment can be filled with air, agas, or a liquid such as a solution or water. The ‘x’ marks on thesurface of 80 facing the sample compartment emphasize that the sample ofinterest (eg., fabricated probes) are placed on this side. Substrate 80can be a ‘chip’ which can be slid out between 75 and 85, allowing formeasurement of different substrates. Element 90 in the drawing refers toa port in the sample compartment for drawing liquid or gases in and outof the compartment, for instance by pumps, electrostatic means, etc. Theentire sample assembly can be mounted on an x-y translation stage 95 ifnecessary.

[0395]FIG. 3 illustrates an embodiment in which a slab-dielectricwaveguide is used to deliver the fundamental light to the sample surface(the light beams are generated, directed and detected as in Drawing Iwith elements 1-5 and 8-13). A parallel plate or dielectric waveguidecan be used to couple the fundamental light into a waveguide propagatingmode. The drawing shows two slabs (110 and 115) and region (120). If theindices of refraction of slab 115 and region 120 are less than the indexof refraction of the light (for both fundamental and second harmonic), awaveguiding mode can be developed. This mode produces multiple internalreflections at the substrate which can be used to increase the amount ofsecond harmonic light generated by the interface. The fundamental beam100 can be coupled into the waveguide 110 using a diffraction grating105 scribed or embossed on the top surface of the waveguide, forexample. The fundamental is propagated along the length of the waveguideand makes multiple total internal reflections at the top and bottomsurfaces. The ‘x’ marks on substrate 110 denote the surface sample to bemeasured (i.e., containing the probes). If this interface generatessignificantly more second harmonic light than the interface betweenmaterials 110 and 115, the light intensity can be neglected. Forexample, if SH-labeled targets are bound to immobilized probes at the‘x’ locations and the atomic structure at the interface between 110 and115 is epitaxially matched, the interface 110/120 will generate muchmore second harmonic light than the interface 110/115.

[0396] In an alternative embodiment, a planar waveguide structure 110 isused for the solid substrate (FIG. 3). In this embodiment, a thin layerof high index of refraction material 115 (the waveguide), such as TiO₂or Ta₂O₅, is deposited on top of the substrate 110 (typically glass). Athin diffraction grating 115 is scribed into this waveguide and lightfrom the laser 100 is coupled using this grating into the waveguide.Second harmonic light can be collected using lenses and filters anddetected with either a PMT-type device or a CCD camera.

[0397] FIGS. 4A-C illustrate an embodiment of a flow cell for carryingout probe-target reactions. The flow cell is 3220 is shown in detail.FIG. 4A is a front view, FIG. 4B is a cross sectional view, and FIG. 4Cis a back view of the cavity. Referring to FIG. 4A, flow cell 3220includes a cavity 3235 on a surface 4202 thereon. The depth of thecavity, for example, may be between about 10 and 1500 .mu.m, but otherdepths may be used. Typically, the surface area of the cavity is greaterthan the size of the probe sample, which may be about 13×13 mm. Inletport 4220 and outlet port 4230 communicate with the cavity. In someembodiments, the ports may have a diameter of about 300 to 400 .mu.m andare coupled to a refrigerated circulating bath via tubes 4221 and 4231,respectively, for controlling temperature in the cavity. Therefrigerated bath circulates water at a specified temperature into andthrough the cavity.

[0398] A plurality of slots 4208 may be formed around the cavity tothermally isolate it from the rest of the flow cell body. Because thethermal mass of the flow cell is reduced, the temperature within thecavity is more efficiently and accurately controlled.

[0399] In some embodiments, a panel 4205 having a substantially flatsurface divides the cavity into two subcavities. Panel 4205, forexample, may be a light absorptive glass such as an RG1000 nm long passfilter. The high absorbance of the RG1000 glass across the visiblespectrum (surface emissivity of RG1000 is not detectable at anywavelengths below 700 nm) substantially suppresses any backgroundluminescence that may be excited by the incident wavelength. Thepolished flat surface of the light-absorbing glass also reducesscattering of incident light, lessening the burden of filtering straylight at the incident wavelength. The glass also provides a durablemedium for subdividing the cavity since it is relatively immune tocorrosion in the high salt environment common in DNA hybridizationexperiments or other chemical reactions.

[0400] Panel 4205 may be mounted to the flow cell by a plurality ofscrews, clips, RTV silicone cement, or other adhesives. Referring toFIG. 4B, subcavity 4260, which contains inlet port 4220 and outlet port4230, is sealed by panel 4205. Accordingly, water from the refrigeratedbath is isolated from cavity 3235. This design provides separatecavities for conducting chemical reaction and controlling temperature.Since the cavity for controlling temperature is directly below thereaction cavity, the temperature parameter of the reaction is controlledmore effectively.

[0401] Substrate 130 is mated to surface 4202 and seals cavity 3235.Preferably, the probe array on the substrate is contained in cavity 3235when the substrate is mated to the flow cell. In some embodiments, anO-ring 4480 or other sealing material may be provided to improve matingbetween the substrate and flow cell. Optionally, edge 4206 of panel 4205is beveled to allow for the use of a larger seal cross section toimprove mating without increasing the volume of the cavity. In someinstances, it is desirable to maintain the cavity volume as small aspossible so as to control reaction parameters, such as temperature orconcentration of chemicals more accurately. In additional, waste may bereduced since smaller volume requires smaller amount of material toperform the experiment.

[0402] Referring back to FIG. 4A, a groove 4211 is optionally formed onsurface 4202. The groove, for example, may be about 2 mm deep and 2 mmwide. In one embodiment, groove 4211 is covered by the substrate when itis mounted on surface 4202. The groove communicates with channel 4213and vacuum fitting 4212 which is connected to a vacuum pump. The vacuumpump creates a vacuum in the groove that causes the substrate to adhereto surface 4202. Optionally, one or more gaskets may be provided toimprove the sealing between the flow cell and substrate.

[0403]FIG. 4D illustrates an alternative technique for mating thesubstrate to the flow cell. When mounted to the flow cell, a panel 4290exerts a force that is sufficient to immobilize substrate 130 locatedtherebetween. Panel 4290, for example, may be mounted by a plurality ofscrews 4291, clips, clamps, pins, or other mounting devices. In someembodiments, panel 4290 includes an opening 4295 for exposing the sampleto the incident light. Opening 4295 may optionally be covered with aglass or other substantially transparent or translucent materials.Alternatively, panel 4290 may be composed of a substantially transparentor translucent material.

[0404] In reference to FIG. 4A, panel 4205 includes ports 4270 and 4280that communicate with subcavity 3235. A tube 4271 is connected to port4270 and a tube 4281 is connected to port 4280. Tubes 4271 and 4281 areinserted through tubes 4221 and 4231, respectively, by connectors 4222.Connectors 4222, for example, may be T-connectors, each having a seal4225 located at opening 4223. Seal 4225 prevents the water from therefrigerated bath from leaking out through the connector. It will beunderstood that other configurations, such as providing additional portssimilar to ports 4220 and 4230, may be employed.

[0405] Tubes 4271 and 4281 allow selected fluids to be introduced intoor circulated through the cavity. In some embodiments, tubes 4271 and4281 may be connected to a pump for circulating fluids through thecavity. In one embodiment, tubes 4271 and 4281 are connected to anagitation system that agitates and circulates fluids through the cavity.

[0406] Referring to FIG. 4C, a groove 4215 is optionally formed on thesurface 4203 of the flow cell. The dimensions of groove, for example,may be about 2 mm deep and 2 mm wide. According to one embodiment,surface 4203 is mated to the translation stage. Groove 4211 is coveredby the translation stage when the flow cell is mated thereto. Groove4215 communicates with channel 4217 and vacuum fitting 4216 which isconnected to a vacuum pump. The pump creates a vacuum in groove 4215 andcauses the surface 4203 to adhere to the translation stage. Optionally,additional grooves may be formed to increase the mating force.Alternatively, the flow cell may be mounted on the translation stage byscrews, clips, pins, various types of adhesives, or other fasteningtechniques.

[0407] In a further alternative embodiment, a suspension of beads,cells, liposomes or other objects comprise the probes (130) as shown inFIGS. 5A-C. The scattered nonlinear light from such a sample—eg., anisotropic sample in which each individual beads or other objects areabout a coherence length or farther apart—is generated in variousdirections with some distribution in intensity. Fundamental light istransmitted through the suspension (130) and the nonlinear radiationcollected. A number of modes of collecting the scattered nonlinear lightis available. For example, collection of the second harmonic can be inthe forward direction (A), at a right angle to the fundamental light(B), or using an integrating sphere approach (C). Part C shows anintegrating sphere 165 with the sample 150 placed inside. Fundamentallight (145) enters the entrance port (170), passes through the sample(150), undergoes a reflection at the sphere wall, and is stopped bybaffle (175). The scattered second harmonic light is collected from thesphere surface through exit port (155) and coupled out of the sphere bya fiber optic line (160). Cells are a convenient and natural system ofstudy for conformational changes in receptors, cell surface moleculesand other biological components. Beads can support phospholipid bilayers(eg., with membrane proteins) or probes such as proteins or nucleicacids can be attached to their surface. The beads provide a large amountof distributed surface area in the sample and can be a usefulalternative to planar surface geometries, especially when thefundamental and nonlinear light is used in the transmission mode.

[0408] In an alternative embodiment (FIG. 6), the excitation light istranformed from a point-like shape into some other shape using variousoptics. For instance, the point-like beam shape of the fundamental beamcan be transformed into a line shape, useful for scanning the samplesurface. However, because the intensity of the nonlinear beam dependson, among other factors, the intensity of the fundamental (typically aquadratic dependence on the fundamental intensity), this transformationwill result in less nonlinear light intensity generated at a givenlocation. To generate a line-shape in the fundamental (which cantypically be a round point of ˜2 mm diameter), one can direct thefundamental beam into a microscope objective which has a magnificationpower of about 10 followed by a 150 mm achromat to collimate the beam aswell known in the prior art and as disclosed in detail in U.S. Pat. No.5,834,758. As shown in FIG. 6, the fundamental light 180 is a beam oftypically 2-3 mm diameter. This beam is directed through a microscopeobjective 185. The objective, which has a magnification power of 10,expands the beam to about 30 mm. The beam then passes through a lens190. The lens, which can be a 150 mm achromat, collimates the beam.Typically, the radial intensity of the expanded collimated beam has aGaussian profile. To minimize intensity variations in the beam, a mask195 can be inserted after lens 190 to mask the top and bottom of thebeam, thereby passing only the central portion of the beam. In oneembodiment, the mask passes a horizontal band that is about 7.5 mm.Thereafter, the beam passes through a cylindrical lens 200 having ahorizontal cylinder axis, which can be a 100 mm f.l. made by MellesGriot. The cylindrical lens expands the beam spot vertically.Alternatively, a hyperbolic lens can be used to expand the beamvertically while resulting in a flattened radial intensity distribution.From the cylindrical lens, the light passes through a lens 205.Optionally, a planar mirror can be inserted after the cylindrical lensto reflect the excitation light toward lens 205. To achieve a beamheight of about 15 mm, the ratio of the focal lengths of the cylindricallens 200 and lens 205 is approximately 1:2, thus magnifying the beam toabout 15 mm. Lens 205, which in some embodiments is a 80 mm achromat,focuses the light to a line of about 15 mm×50 microns at the samplesurface 210.

[0409] In an alternative embodiment shown in FIGS. 7A-B, probes arepatterned in a two-dimensional array (A, top view of array on surface)where each region on the surface—{1,35} in this example—can be adifferent oligonucleotide or protein sequence (or a combination of thesame and different sequences). Part B shows a side-view of the samplesurface (220) in a well (215) containing the targets (225) shown here asprotein objects with second-harmonic-active labels (X) attached. Thewell can hold liquid or buffer and serves to physically separate thecontents of the well from other parts of the substrate or other elementsin a substrate array. The fundamental light can be multiplexed and eachresultant beam can be guided by individual mirrors to simultaneouslyscan different lines or regions within the array, thus increasing evenfurther the potential of the technique for high-throughput studies.

[0410] In an alternative embodiment, the method of Levicky et al. “UsingSelf-Assembly to Control the Structure of DNA Monolayers on Gold: ANeutron Reflectivity Study,” Journal of the American Chemical Society120: 9787-9792 (1998), or the method of Chrisey et al., “CovalentAttachment of Synthetic DNA to Self-Assembled Monolayer Films”, NucleicAcids Research 24, 3031, (1996), is used to attach the probe DNA to thesubstrate. In the method of Chrisey et al., as illustrated in FIG. 8, afused silica or oxidized silicon substrate is used (230) and derivatizedwith N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (EDA) (235). In oneembodiment, the EDA-modified surface is then treated with theheterobifunctional crosslinker (SMPB), whose succinimide ester moietyreacts with the primary amino group of EDA (240). A thiol-DNA oligomersubsequently (245) of base-pair sequence (xzzy) (where ‘xzzy’ representsthe entire sequence) reacts with the maleimide portion of the SMPBcrosslinker, to yield the covalently bound species shown (250).

[0411] In an alternative embodiment, elements in the surface array arephysically separated as illustrated in FIG. 9, allowing for differenttargets, target solutions, etc. to be added selectively to any or all ofthe elements. Part (A) is a top-view of the substrate (255) withpartitions or walls (260) separating the different well regions—in thisexample, 16 wells. Part (B) shows a side-view of a well (265) withattached probes (270). Such arrays are commonly found in the art, suchas the 96-well plates, etc. and are commercially available (FisherScientific, Inc. etc.)

[0412] In an alternative embodiment, a glass substrate surface can becoated with a layer of a reflective metal such as silver. The metalliclayer will increase nonlinear optical generation and collection.Biomolecules or other particles can be attached to derivatized layersbuilt on top of the metal. For instance, the metal can be coated with alayer of silicon dioxide (SiO₂), then with a layer of aminosilane suchas 3-amino-octyl-trimethoxysilane. Oligonucleotides or polynucleotidescan then be attached to the aminosilane layer using linkers whichconnect the 3′ or 5′ end of the oligo to the amine group. Alternatively,the oligos or polynucleotides can be adsorbed to the aminosilane layer.FIG. 10 illustrates an embodiment of this type where a glass substrate(275) is derivatized with a Ag layer (280). A thin coat of SiO₂ is thendeposited on top of the silver layer (285) and derivatized with theaminosilane (290).

[0413] In a specific embodiment, nucleic acid or PNA microarrays can beobtained commercially or constructed according to public literature(eg., http://cmgm.stanford.edu/pbrown/mguide/index.html). The surfacechemistry to be used is that found in Chrisey et al., “CovalentAttachment of Synthetic DNA to Self-Assembled Monolayer Films”, NucleicAcids Research 24, 3031, (1996), in which oligonucleotides are attachedto self-assembled monolayer silane films on fused silica slides.Silanization is accomplished viaN-(2-aminoethyl)-3-aminopropyltrimethoxysilane.

[0414] In other embodiments, oligonucleotides or PNAs can be attached tothe solid substrate via light-directed synthesis (S. A. Fodor, Science277 (1997), 393) or via chemical synthesis (e.g., Chrisey et al.,“Covalent Attachment of Synthetic DNA to Self-Assembled MonolayerFilms”, Nucleic Acids Research 24, 3031, (1996)).

[0415] In still other embodiments, surfaces or microarrays microarraysof oligonucleotides or PNAs can be obtained commercially or constructedaccording to public literature (eg.,http://cmgm.stanford.edu/pbrown/mguide/index.html).

[0416] DNA microarrays can be obtained commercially or constructed, forexample, according to public literature (e.g.,http://cmgm.stanford.edu/pbrown/mguide/index.html). The surfacechemistry preferably to be used is that found in Chrisey et al.,“Covalent Attachment of Synthetic DNA to Self-Assembled MonolayerFilms”, Nucleic Acids Research 24, 3031, (1996), in whicholigonucleotides are attached to self-assembled monolayer silane filmson fused silica slides. Silanization is done viaN-(2-aminoethyl)-3-aminopropyltrimethoxysilane.and Hoheisel, J. D.“Improved solid supports and spacer/linker systems for the synthesis ofspatially addressable PNA-libraries” Nucleosides Nucleotides 18 (1999)1289-1291 on glass or silica. The buffer or solution in contact with thePNA oligonucleotides can be chosen from a range of those known in theart. Hybridization and wash solutions are found in the art. For example,the web site: cmgm.stanford.edu/pbrown/protocols gives detailedinstructions for probe-target hybridization.

[0417] Microarrays can be mounted on an x-y translation stage and drivenby personal computer (PC control) using a motorized translator (acquiredfrom Oriel, Inc.) or using one of the many procedures in the art (e.g.,V. G. Cheung et al., “Making and reading microarrays”, Nature Genetics(Suppl.), 1999, 21: 15-19).

[0418] In an alternative embodiment, bead-based fiber-optic arrays canbe used (ref. 34) in which light beams (eg., fundamental and secondharmonic) travel via total internal reflection along the path of thefiber. The fundamental light is coupled into the bundle or individualoptical fibers and second harmonic light is generated at the tip surfaceand collected back through the fiber. FIGS. 11A-B illustrate afiber-optic bundle array. Part (A) shows a bundle of fiber optic cables(295) with wells at the distals ends for placement of beads (300). Part(B) shows a close-up view of a single optical fiber. Fundamental lighttravels (ω) toward the distal end with the bead (305). Some fundamentallight is scattered back from the bead along with second harmonic light(2ω) and travels back through the fiber to the proximal end where anoptical train and detection system (not shown) separates the fundamentalradiation from the second harmonic radiation. Bead (310) is covered withprobes.

[0419] In an alternative embodiment, green fluorescent protein (GFP) ornonlinear-active mutants thereof are used to label proteins in-vivo viamutagenesis according to procedures well known in the art. The GFP ormutants thereof are second-harmonic active and can serve as a built-inlabel of proteins. For example: a cell membrane receptor can be labeledwith GFP via mutagenesis. Cells containing this GFP-tagged receptorproduce some background second harmonic light when illuminated with afundamental beam in a surface-selective nonlinear optical technique.When ligands or other compounds which bind to the receptor and induceactivation—and a concomitant conformational change—the intensity and/orspectrum and/or time-course of the second harmonic light will change(due, for example, to a change in overall net orientation of the GFPprotein) as the GFP label moves slightly due to the conformationalchange on the receptor. The change in measured nonlinear opticalproperties are thus correlated with conformational change and ligandbinding.

[0420] In an alternative embodiment, the detector (65) of the nonlinearradiation in FIG. 1 is a photomultiplier tube operated in single-photoncounting mode. Pbotocurrent pulses can be voltage converted, amplified,subjected to discrimination using a Model SR445 Fast Preamplifier andModel SR 400 Discriminator (supplied by Stanford Research Systems, Inc.)and then sent to a counter (Model 3615 Hex Scaler supplied by KineticSystems). Photon counter gating and galvo control through a DAC output(Model 3112, 12-Bit DAC supplied by Kinetic Systems) can be synchronizedusing a digital delay/pulse generator (Model DG535 supplied by StanfordResearch Systems, Inc.). Communication with a PC computer 29 can beaccomplished using a parallel register (Model PR-604 supplied by DSPTechnologies, Inc.), a CAMAC controller card (Model 6002, supplied byDSP Technologies, Inc.) and a PC adapter card (Model PC-004 supplied byDSP Technologies, Inc.).

[0421] In an alternative embodiment, a bandpass, notch, or color filteris placed in either or all of the beam paths (eg., fundamental, secondharmonic, etc.) allowing, for example, for a wider spectral bandwidth ormore light throughput.

[0422] In an alternative embodiment, an interference, notch-pass,bandpass, reflecting, or absorbant filter can be used in place of thefilters in the figures in order to either pass or block the fundamentalor nonlinear optical beams.

[0423] According to another embodiment, detection of the nonlinearoptical light is achieved using a charge coupled detector (CCD) in placeof a photomultiplier tube or other photodetector. The CCD subsystemcommunicates with and is controlled by a data acquisition boardinstalled in a computer. Data acquisition board may be of the type thatis well known in the art such as a CIO-DAS16/Jr manufactured by ComputerBoards Inc. The data acquisition board and CCD subsystem, for example,may operate in the following manner. The data acquisition board controlsthe CCD integration period by sending a clock signal to the CCDsubsystem. In one embodiment, the CCD subsystem sets the CCD integrationperiod at 4096 clock periods. by changing the clock rate, the actualtime in which the CCD integrates data can be manipulated. During anintegration period, each photodiode accumulates a charge proportional tothe amount of light that reaches it. Upon termination of the integrationperiod, the charges are transferred to the CCD's shift registers and anew integration period commences. The shift registers store the chargesas voltages which represent the light pattern incident on the CCD array.The voltages are then transmitted at the clock rate to the dataacquisition board, where they are digitized and stored in the computer'smemory. In this manner, a strip of the sample is imaged during eachintegration period. Thereafter, a subsequent row is integrated until thesample is completely scanned.

[0424] In a specific embodiment, the nonlinear spectrum of a sample ismeasured by measuring the nonlinear radiation (e.g., second harmonicradiation) at two or more spectral points or bands, using amonochromator, filter or other wavelength-selecting device to accomplishthis.

[0425] In a specific embodiment, a monochromator (60) can be placedbefore the detecting element in the device, in order to spectrallyresolve the nonlinear optical radiation (FIG. 1).

[0426] In a specific embodiment, imaging techniques described in the art(Peleg et al., “Nonlinear optical measurement of membrane potentialaround single molecules at selected cellular sites,” Proc. Natl. Acad.Sci. V. 96, 1999, 6700-6704, or Campagnola et al., “High-resolutionnonlinear optical imaging of live cells by second harmonic generation,”Biophysical Journal 77 (6), 3341-3349 (1999), can be performed usingSHG-labeled components (such as labeled ligands or receptors) instead ofthe membrane-intercalating dyes used in the art. These imagingtechniques can be used to image solid surfaces, cell surfaces or otherinterface using SHG-labeled components.

[0427] In a specific embodiment, channels (or microfluid) channels canbe used to introduce the components into the sample cell via positivedisplacement, pumping, electrophoretic means or other means known in theart for manipulating the flow of components into and out of a reactionchamber.

[0428] In a specific embodiment, the apparatus can be assembled into auser-closed product with a user-controlled interface (an LED panel, forexample, or PC-based software) with the option of inserting and removingdisposable substrates (e.g., biochips) with the attached probes.

[0429] In a specific embodiment, a photodiode, avalanche photodiode orother photoelectric detector (65) in FIG. 1 is used as the lightdetection means.

[0430] In a specific embodiment, a surface array can be used that is ina fixed position and the incident light beam scanned across the surfaceusing methods well known in the art, such as a galvanometer mirror or apolygonal mirror.

[0431] An alternative embodiment comprises a scanning or imaging methodwhere a physical property of the nonlinear radiation is measured asfunction of position (x,y,z) in a sample. The scanning method cancomprise a combination of both stage translation (x-y) and beamscanning, wherein, for example, the latter controls the incidentposition of the fundamental beam on the array surface.

[0432] In a specific embodiment, a stop-flow mixing chamber is used torapidly mix the components in the sample cell.

[0433] In a specific embodiment, the proportionality constant(calibration curve of intensity of second harmonic light vs.concentration of targets bound to attached probes) is determined bymeasuring the concentration of targets using another method such asradiolabeling or fluorescence labels of the targets. Once thecalibration curve is known, for a given probe and target type (e.g.,cDNA, RNA, size of oligos, etc.), the concentration of bound target isdetermined using this relation and the measured second harmonicintensity. This embodiment can be generalized to any other nonlinearlight beam emanating from the sample, including third harmonic, sum ordifference frequency light.

[0434] In a specific embodiment, the nonlinear optical,surface-selective apparatus can comprise a unit without the lightexcitation source (e.g., with sample compartment, filters, detectors,monochromator, computer interface, software, or other parts) so that theuser can supply his own excitation source and adapt its use to themethods described herein.

[0435] In an alternative embodiment, measurable information can berecorded in real time.

[0436] Various Configurations of an Apparatus Using theSurface-Selective Nonlinear Optical Technique in the Present Invention.

[0437] The apparatus for detection of the probe-target reactions ortheir effects can assume a variety of configurations. In its most simpleform, the apparatus will comprise the following:

[0438] i) a source of the fundamental light

[0439] ii) a detector for measuring the intensity of the second harmonicor other nonlinear optical beams.

[0440] More elaborate versions of the apparatus will employ, forexample: a monochromator (for wavelength selection), a pass-filter,color filter, interference or other spectral filter (for wavelengthselection or to separate the fundamental(s) from the higher harmonics),one or more polarizing optics, a means of applying an electric field,one or more mirrors or lenses for directing and focusing the beams,computer control, software, etc.

[0441] The mode of delivering or generating the nonlinear optical light(e.g., SHG) can be based on one or more of the following means: TIR(Total internal reflection), Fiber optics (with or without attachedbeads), Transmission (fundamental passes through the sample), Reflection(fundamental is reflected from the sample), scanning imaging (allows oneto scan a sample), confocal imaging or scanning, resonance cavity forpower build-up, multiple-pass set-up.

[0442] Measured information can take the form of a vector which caninclude one or more of the following parameters: {intensity of light(typically converted to a photovoltage by a PMT or photodiode),wavelength of light (determined with a monochromator and/or filters),time, substrate position (for array samples, for instance, wheredifferent sub-samples are encoded as function of substrate location andthe fundamental is directed to various (x,y) locations}. Two generalconfigurations of the apparatus are: image scanning (imaging of asubstrate—intensity, wavelength, etc. as a function of x,y coordinate)and spectroscopic (measurement of the intensity, wavelength, etc. forsome planar surface or for a suspension of cells, liposomes or otherparticles).

[0443] The fundamental beam can be delivered to the sample in a varietyof ways. FIGS. 12-16 are schematics of various modes of delivering thefundamental and generating second harmonic beams. It is understood thatin sum- or difference-frequency configurations, the fundamental beamswill be comprised of two or more beams, and will generate, at theinterfaces, the difference or sum frequency beams. For the purposes ofillustration, only the second harmonic generation case is described indetail herein. Furthermore, it shall be understood that the sample cell3 in all cases can be mounted on a translation stage (1-, 2-, or3-dimensional degrees of freedom) for selecting precise locations of theinterfacial interaction volume. The sample cell in all cases can befitted with flow ports and tubes which can serve to introduce (or flushout) components such as molecules, particles, cells, etc.

[0444] Transmission

[0445]FIG. 12A is a schematic of a configuration relying on transmissionof the fundamental and second harmonic beams. The fundamental 320 (ω)passes through the sample cell 330 and interacts within a volume element(denoted by the circle) in which are contained one or more interfacescapable of generating the second harmonic beam 325 (2ω). The fundamentaland second harmonic beams are substantially co-linear as denoted by beam325. The sample cell can contain suspended beads, particles, liposomes,biological cells, etc. in some medium, providing interfacial areacapable of generating second harmonics in response to the fundamentalbeam. As shown, the second harmonic is detected co-linearly with thefundamental direction, but could alternatively be detected off-anglefrom the fundamental, for instance at 90° to the fundamental beam.

[0446]FIG. 12B is a schematic of another configuration relying ontransmission of the fundamental and second harmonic beams. Thefundamental 335 is directed onto a sample cell 345 and the secondharmonic waves are generated at the top surface—this surface can bederivatized with immobilized probes or with adsorbed particles,liposomes, cells, etc. The second harmonic waves 340 are generatedwithin a volume element denoted by the circle at the interface betweenthe top surface and the medium contained within cell.

[0447]FIG. 12C is a schematic of a configuration substantially similarto the one depicted in FIG. 2A except that the bottom surface of thesample cell 3, rather than the top, is used to generate the secondharmonic waves.

[0448] Total Internal Reflection

[0449]FIG. 13A is a schematic of a waveguide 4 capable of acting as atotal internal reflection waveguide which refracts the fundamental 365and directs it to a location at the interface between the waveguide 380and a sample cell 375. At this location, denoted by the circle, thefundamental will generate the second harmonic waves and undergo totalinternal reflection; the second harmonic beam will propagatesubstantially colinearly with the fundamental and exit the prism 380.Waveguide 380 will typically be in contact with air. In thisillustration, the waveguide 380 is a Dove prism.

[0450]FIG. 13B is a schematic of a configuration similar to the onedepicted in FIG. 13A except that the waveguide 400 allows for multiplepoints of total internal reflection between the waveguide 4 and thesample cell 395, increasing the-amount of second harmonic lightgenerated from the fundamental beam.

[0451] Fiber Optic

[0452]FIG. 14 depicts various configurations of a fiber optic means ofdelivering or collecting the fundamental or second harmonic beams. InFIG. 14A, the coupling element 410 between a source of the fundamentalwave and the fiber optic is depicted. The fundamental, thus coupled intothe fiber optic waveguide 405, proceeds to a sample cell 415. In FIG.14A, the tip of the fiber can serve as the interface of interest capableof generating second harmonic waves, or the tip can serve merely tointroduce the fundamental beam to the sample cell containing suspendedcells, particles, etc. In FIG. 14A, the second harmonic light iscollected back through the fiber optic.

[0453]FIG. 14B is identical to FIG. 14A except that a bead is attachedto the tip of the fiber optic (according to means well known in theart). The bead can serve to both improve collection efficiency of thesecond harmonic light or be derivatized with probes or adsorbed speciesand presenting an interface with the medium of sample cell 425 capableof generating the second harmonic light.

[0454]FIG. 14C is identical to both FIGS. 14A and 14B except thatcollection of the second harmonic light is effected using a solid-angledetector 450.

[0455] Optical Resonance Cavity

[0456] An optical resonance cavity is defined between at least tworeflective elements and has an intracavity light beam along anintracavity beam path. The optical cavity or resonator consists of twoor more mirrored surfaces arranged so that the incident light can betrapped bouncing back and forth between the mirrors. In this way, thelight inside the cavity can be many orders of magnitude more intensethan the incident light. This phenomenon is well known and has beenexploited in various ways (see, for example, Yariv A. “Introduction toOptical Electronics”, 2^(nd) Ed., Holt, Reinhart and Winston, N.Y. 1976,Chapter 8). The sample cell can be present in the optical cavity or itcan be outside the optical resonance cavity.

[0457]FIG. 15 is a schematic of an optical resonance power build-upcavity configuration. FIG. 15A is a schematic of an optical resonancecavity in which the sample cell 465 is positioned intracavity and thefundamental and second harmonic beams are transmitted through it—auseful configuration for sample cells containing suspended particles,cells, beads, etc. The fundamental beam 455 enters the optical resonancecavity at reflective optic 460 and builds up in power between reflectiveelements 460 and 462 (intracavity beam). Mirror 460 is preferably tilted(not perpendicular to the direction of the incident fundamental 455) toprevent direct reflection of the intracavity beam back into the lightsource. The natural reflectivity and transmisivity of 460 and 462 can beadjusted so that the fundamental builds up to a convenient level ofpower within the cavity. The fundamental generates second harmonic lightin a volume element within the sample cell denoted by the circle.Reflective optic 460 can reflect the fundamental and the secondharmonic, while reflective optic 462 will substantially reflect thefundamental but allow the pass-through of the second harmonic beam 475which is subsequently detected. U.S. Pat. No. 5,432,610 (King et al.)describes a diode-pumped power build-up cavity for chemical sensing andit and the references it makes are hereby incorporated by referenceherein.

[0458]FIG. 15B is a schematic of an optical resonance power build-upcavity configuration in which the fundamental beam 475 enters theoptical cavity by reflection from optic 480. A second reflective opticelement 482 defines the optical resonance cavity. Element 490 is awaveguide (such as a prism) in contact with the sample cell 485 andallows total internal reflection of the fundamental beam at theinterface between the waveguide and sample cell surfaces, generating thesecond harmonic light. Element 482 substantially reflects thefundamental beam but passes through the second harmonic beam 495 whichis subsequently detected.

[0459] Reflection

[0460]FIG. 16A is a schematic of a configuration involving reflection ofthe fundamental and second harmonic beams. A substrate 525 is coatedwith a thin layer of a reflective material 520, such as a metal, and ontop of this is deposited at layer 515 suitable for attachment of theprobes or adsorption of particles, cells, etc. (e.g., SiO₂). This layeris in contact with the sample cell 510. The fundamental 500 passesthrough the sample cell 510 and generates a second harmonic wave at theinterface between layers 515 and 520. The fundamental and secondharmonic waves 505 are reflected back from the surface of layer 520.

[0461]FIG. 16B is substantially similar to FIG. 15A except that thesecond harmonic and fundamental beams are reflected 535 from theinterface between the medium contained in sample cell 540 and layer 545.Layer 545 is reflective or partly reflective layer deposited onsubstrate 550 and is suitable for adsorption of particles, cells, etc.or attachment of probes.

[0462]FIG. 16C is a schematic illustrating that only the sample cell 565need be used for a reflective geometry. The sample cell 565 is partlyfilled with some medium 570 and the fundamental and second harmonicbeams are reflected 560 from the gas-liquid or vapor-liquid interface atthe surface of 570.

[0463] Modes of Detection

[0464] Charge-coupled detectors (CCD) array detectors can beparticularly useful when information is desired as a function ofsubstrate location (x,y). CCDs comprise an array of pixels (i.e.,photodiodes), each pixel of which can independently measuring lightimpinging on it. For a given apparatus geometry, nonlinear light arisingfrom a particular substrate location (x,y) can be determined bymeasuring the intensity of nonlinear light impinging on a CCD arraylocation (Q,R) some distance from the substrate—this can be determinedbecause of the coherent, collimated (and generally co-propagating withthe fundamental) nonlinear optical beam) compared with the spontaneous,stochastic and multidirectional nature of fluorescence emission. With aCCD array, one or more array elements {Q,R} in the detector will map tospecific regions of a substrate surface, allowing for easy determinationof information as a function of substrate location (x,y). Photodiodedetector and photomultiplier tubes (PMTs), avalanche photodiodes,phototransistors, vacuum photodiodes or other detectors known in the artfor converting incident light to an electrical signal (i.e., current,voltage, etc.) can also be used to detect light intensities. For CCDdetector, the CCD communicates with and is controlled by a dataacquisition board installed in the apparatus computer. The dataacquisition board can be of the type that is well known in the art suchas a CIO-DAS16/Jr manufactured by Computer Boards Inc. The dataacquisition board and CCD subsystem, for example, can operate in thefollowing manner. The data acquisition board controls the CCDintegration period by sending a clock signal to the CCD subsystem. Inone embodiment, the CCD subsystem sets the CCD intregration period at4096 clock periods. By changing the clock rate, the actual time in whichthe CCD integrates data can be manipulated. During an integrationperiod, each photodiode accumulates a charge proportional to the amountof light that reaches it. Upon termination of the integration period,the charge is transferred to the CCD's shift registers and a newintegration period commences. The shift registers store the charges asvoltages which represent the light pattern incident on the CCD array.The voltages are then trasmitted at the clock rate to the dataacquisition board, where they are digitized and stored in the computer'smemory. In this manner, a strip of the sample is imaged during eachintegration period. Thereafter, a subsequent row is integrated until thesample is completely scanned.

[0465] Sample Substrates and Sample Cells

[0466] Sample substrates and cells can take a variety of forms drawingfrom, but not limited to, one or more of the following characteristics:fully sealed, sealed or unsealed and connected to flow cells and pumps,integrated substrates with a total internal reflection prism allowingfor evanescent generation of the nonlinear beam, integrated substrateswith a resonant cavity for fundamental power build-up, an optical set-upallowing for multiple passes of the fundamental for increased nonlinearresponse, sample cells containing suspended biological cells, particles,beads, etc.

[0467] Data Analysis

[0468] Data analysis operates on the vectors of information measured bythe detector. The information can be time-dependent and kinetic. It canbe dependent on the concentration of one or more biological components,inhibitors, antagonists, agonists, drugs, small molecules, etc. whichcan be changed during a measurement or between measurements. It can alsobe dependent on wavelength, etc. In general, the intensity of nonlinearlight will be transformed into a concentration or amount of a particularstate (for example, the surface-associated concentration of a componentor the amount of opened or closed ion-channels in cell membranes) viathe detected change in nonlinear optical properties that are correlatedto conformational changes induced in the sample.

[0469] Details of the data analysis can vary from experiment toexperiment. There is a large literature available for makingcorrelations between conformation and fluorescence intensity (see thereferences by Glauner et al., Nature 402, 813 (1999), Ghanouni et al.,Proc. Natl.. Acad. Sci., v.98, 5997 (2001), and Ghanouni et al., Journalof Biological Chemistry, v.276, 24433 (2001), and references therein).Analogous procedures are constructed for the nonlinear opticaltechniques. For instance, the square root of the intensity of secondharmonic light (proportional to electric field amplitude of the light)is proportional to the number of nonlinear-active species in a sampletimes the orientational average of the hyperpolarizability of thespecies. This is a well known relationship that can be used to quantifyconformational change (and in turn binding affinity to a probe) withintensity of a nonlinear beam. Kinetics and equilibrium properties ofthe reactions of interest can be determined via the measurements andappropriate data analysis.

[0470] Screening for Candidate Binding Partners

[0471] Candidate binding partners for binding a test molecule can bescreened through the detection of conformational changes on probe-targetbinding. The method of screening one or more candidate binding partnersfor binding to a test molecule involves measuring the one or morephysical properties of the one or nonlinear optical light beamsemanating from said sample comprising the test molecule and the one ormore candidate binding partners, where a change in the one or morephysical properties of the nonlinear light beams relative to a valuemeasured in the absence of exposure to the one or more candidate bindingpartners is an indication of a binding event having occurred. In apreferred embodiment the candidate binding partner is not attached tothe surface.

[0472] The probes or targets of the present invention that can be usedinclude but are not limited to naturally occurring, artificiallyaltered, or genetically engineered, biological species or non-biologicalspecies. The candidates for probes or targets also include but are notlimited to one or more of the following components: a nucleic acid,protein, small molecule, organic molecule, biological cell, virus,molecular beacon, liposome, receptor, antibody, agonist, antagonist,inhibitor, hapten, ligand, antigen, oocyte, hormone, protein, peptide,receptor, drug, lipid, ganglioside, enzyme, nucleotide, carbohydrate,cDNA, oligonucleotide, nucleoside, polynucleoside, polynucleotide,lipid, ganglioside, oligosaccharide, peptide nucleic acid (PNA), toxin,nucleic acid analog, ion channel receptor, G coupled-protein receptor.In a specific embodiment, the probes can be patterned in an array formaton a substrate or solid surface, with the properties or chemicalidentity of the probes remaining constant or varying among regions ofthe array.

[0473] In one embodiment of the selection of candidate binding partners,an external electric field can be applied to the samples to create thenon-centrosymmetric condition required for the nonlinear opticaltechniques. For example, proteins (e.g., solubilized GPCRs) that arelabeled with a nonlinear-active label can be partially oriented in theelectric field. A background nonlinear light signal is measured. When aligand that binds to the protein is added to the medium, it triggers aconformational change in the protein (well known to those skilled in theart) that results in a change in the properties of the nonlinear lightsignal (e.g., a change in intensity). This scheme can be very useful inscreening libraries of ligands (small molecules, drugs, etc.) forbinding to proteins—i.e., high-throughput drug screening. In a preferredembodiment, the proteins are GPCRs, a well known as a class of proteinsthat are implicated in disease, and for which drugs can be developed.Drugs can be agonists, antagonists, inhibitors, etc. that interact(e.g., bind) with the receptors in some way. The following references(and references therein) describe different exemplary GPCRs that can beused:

[0474] P. Ghanouni et al., “Agonist-induced conformational changes inthe G-protein-coupling domain of the β₂ adrenergic receptor”, Proc.Natl.. Acad. Sci., v.98, 5997 (2001).

[0475] P. Ghanouni et al., “Functionally Different Agonists InduceDistinct Conformations in the G Protein Coupling Domain of the β₂Adrenergic Receptor”, Journal of Biological Chemistry, v.276, 24433(2001).

[0476] C. Bieri et al., “Micropatterned immobilization of a Gprotein-coupled receptor and direct detection of G protein activation”,Nature Biotechnology, 17, 1105 (1999).

[0477] Helmreich, E. J. M. and Hofmann, K P, “Structure and Function ofProteins in G-protein-coupled signal transfer”, Biochimica et BiophysicaActa 1286, 285 (1996).

[0478] Gether et al. (1995) J Biol Chem 270, 28628-28275, Fluorescentlabeling of purified .beta..sub.2 Adrenergic receptor.

[0479] Turcatti et al. (1996) J Biol Chem 271, 19991-19998, Probing theStructure and Function of the Tachykinin Neurokinin-Receptor throughBiosynthetic incorporation of flourescent amino acids at specific sites.

[0480] Liu et al. (1996) Biochemistry 35, 11865-11873, Site-Directedfluorescent labeling of P-glycoprotein on cysteine residues in thenucleotide binding domains.

[0481] Lodish, Harvey, et al., Molecular Cell Biology (₄ ^(th) ed.,2000).

[0482] Screening for Modulators

[0483] The invention provides a method of screening one or morecandidate modulator molecules for the ability to modulate theinteraction between a test molecule and its binding partner. The actionof modulator and inhibitor molecules have been previously described. Anychange in the one or more physical properties of the nonlinear lightbeam emanating from a sample comprising the test molecule, its bindingpartner and the modulator, relative to what was measured in the absenceof exposure of the test molecule to the binding partners, serves as anindicator of the ability of the candidate modulator molecules tomodulate the interaction between the test molecule and its bindingpartner (i.e. to increase or decrease their binding).

[0484] The invention can be used, for example, to monitor geneexpression or for studies involving drug screening or high-throughputscreening where a candidate drug is tested for binding, or its effect onprobe-target binding, i.e., to reduce or enhance probe-target binding.In other cases, for example, a drug can be tested for efficacy by itsability to bind to a receptor or other molecule on the surface of abiological cell. In another specific embodiment, compounds that arepotential inhibitors of an agonist to a receptor are screened by testingfor blocking of the agonist binding to the receptor, i.e., removal of aconformational change induced by the agonist when the receptor andagonist are also in the presence of an inhibitor candidate.

[0485] In a specific embodiment, the invention is used for drugscreening or high-throughput screening where a candidate drug is testedfor its ability to activate or inhibit a probe (e.g., a receptor, ionchannel protein, etc.). A drug candidate is tested for its ability toactivate a conformational change in a probe—in this case, one seeksagonists of the probe.

[0486] In an alternative embodiment, target-probe interactions can bemeasured in the presence of some modulator of the interactions—themodulator being, for example, a small molecule, drug, or other moiety,molecule or particle which changes in some way the target-probeinteractions (e.g., has some affinity for the probe and blocks orinhibits target binding). The effect of a modulator on probe-targetbinding, where the target is known to bind to the probes, isinvestigated using the nonlinear optical method. The modulator can beadded before, during or after the time in which the probe-targetinteractions occur.

[0487] In a specific embodiment, a biological probe-target bindingreaction can be measured in the presence of agonists, antagonists,drugs, or small molecules which can block, initiate or otherwisemodulate the binding strength (e.g., equilibrium constant) of the saidprobe-target binding reaction. This embodiment can be useful in manycases, for example when one would like to know the efficacy of a drug'sability to block or modulate a certain probe-target reaction for medicaluses or basic research.

[0488] Detection of Conformational Changes

[0489] Conformational changes can be studied by the present invention atan interface or in the bulk, where the conformational change leads to achange in one or more physical properties of one or more nonlinear lightbeams emanating from the sample. The conformational change can beinitiated by a biological or chemical binding event, or by a biologicalcomponent, drug, small molecule, agonist or antagonist binding to amolecule or a particle, and can be assayed as described herein forbinding interactions.

[0490] In a specific embodiment, a change in orientation or dipolemoment occurs in the interfacial region possessing a nonlinearsusceptibility as a result of some probe-target interaction. Anonlinear-active label can be attached to the probe of interest andbinding of the probe to some target (such as a drug candidate tested forits ability to activate the probe and thereby induce a conformationalchange) in solution results in a change in orientation or dipole momentand this changes the nonlinear susceptibility of the interfacial region,and thus the properties of the nonlinear beams (e.g., intensity,polarization, wavelength).

[0491] In another embodiment, a non-centrosymmetric region is created byapplication of an external field (EFISH technique). The region can beinterfacial, bulk or some combination thereof. Probes or targets (theprobes, targets or both are nonlinear-active, either intrinsically orlabeled using a nonlinear-active label) are poled by the electric fieldand this results in a background. When a binding reaction occurs betweenthe probes and targets, this can activate a conformational change in oneor both species or result in a change in dipole moment of species,resulting in a change in the measured nonlinear optical signals.

[0492] For use with nucleic acid hybridization (oligonucleotide,polynucleotide, RNA, etc.), target oligonucleotides can be exposed to asurface of an array on which are situated probe oligonucleotides. At theprobe oligonucleotide sequences in the array (corresponding to knownlocations) where sequence-complementary hybridization and anaccompanying conformational change occurs, the fundamental light wouldgive rise to a change in nonlinear optical signal, or a change in thebackground of such a signal. This can be detected and correlated withthe spatial location of the array element and hence the oligonucleotidesequence. For example, two major applications of nucleic acidmicroarrays are: 1) identification of sequence (gene or genemutation)—monitoring of DNA variations, for example; and 2)determination of expression level (abundance) of genes. There are manyformats that can be used for preparing the arrays. For example, in onecase probe cDNA (500˜5000 base pairs long) can be immobilized to a solidsurface such as glass using robot spotting and exposed to a set oftargets either separately or in a mixture (R. Ekins, F. W. Chu, Trendsin Biotechnology, 1999, 17, 217). Another format involves synthesizingoligonucleotides (20˜25 mer oligos) or peptide nucleic acids probesin-situ (on the solid substrate, Fodor et al., “Light-directed,Spatially Addressable Parallel Chemical Synthesis,” Science 251 (4995):767-773 Feb. 15, 1999) or by conventional synthesis followed by on-chipimmobilization. The array is then exposed to target DNA, hybridized, andthe identity or abundance of complementary sequences are determined.

[0493] Protein arrays can be prepared (see for example, G. MacBeath andS. L. Schreiber, “Printing Proteins as Microarrays for High-ThroughputFunction Determination”, Science 2000, 289, 1760-1763) to determinewhether a given target protein binds to the immobilized probe protein onthe surface. These arrays can be used to study small molecule binding tothe probe proteins.

[0494] Many reviews of microarray technology and applications areavailable in the art. For instance, those of: Ramsay, “DNAchips—states-of-the-art,” Nature Biotechnology 1998, 16(1), 40-44(relevant portions of which are incorporated by reference herein);Marshall et al., “DNA chips—an array of possibilities,” NatureBiotechnology 1998, 16(1), 27-31 (relevant portions of which areincorporated by reference herein); S. A. Fodor, Science 277 (1997), 393(relevant portions of which are incorporated by reference herein); D. H.Duggan et al., Nature Genet. 21 (Suppl.) (1999), 10 (relevant portionsof which are incorporated by reference herein); M. Schena et al.,Science 270 (1995), 467 (relevant portions of which are incorporated byreference herein); L. McAllister et al., Am. J. Hum. Genet. 61 (Suppl.)(1997), 1387 (relevant portions of which are incorporated by referenceherein); and A. P. Blanchard et al., Biosens. And Bioelectron. 11 (1996)687 (relevant portions of which are incorporated by reference herein).

[0495] The conformational changes also allow enable studying the degreeor extent of binding between a probe and a target, utilizing the surfaceselective nonlinear optical techniques, through measuring theconformational effect the binding induces. In a specific embodiment,probes that are labeled with a nonlinear-active label are attached to asolid surface or substrate. When the probes bind to a target, theconformation of the probe changes, and the orientation of the label withrespect to the surface, and fundamental beam changes also. In apreferred embodiment the candidate binding partners (probes) are notattached to the surface. In another embodiment the nonlinear activelabel or moiety is attached to the target or probe molecules in vitro.The degree of probe-target binding can be correlated via the amount ofchange of a measured property of the nonlinear optical radiation (e.g.,intensity). For example, labeled oligonucleotides are attached to asolid substrate according to means well known in the art and exposed totargets to test for hybridization. When hybridization to a targetoccurs, the orientation of the label on the oligonucleotide changes.Microarrays known in the art (varying sequence of oligos in differentsurface locations) or substrates with uniform oligonucleotide sequencescan be used. Binding between surface-attached proteins and targets—otherproteins, ligands, etc. wherein the binding reaction triggers aconformational change are another exemplary embodiment with the presentinvention. In general, any surface-attached species that undergoes aconformational change when binding or interacting with any other speciesor stimulus can be studied with the present invention. Furthermore, thesurface-attached probes need not be labeled when using indicators whichare sensitive to minute changes in electric charge density or changesthereof. If the conformational change in a probe results in a change inthe arrangement of electric charges on a surface—even if this change istransient—indicators near the interface can be used to detect thesechanges and report on the conformational change.

[0496] In a specific embodiment, a MB analogue probe, described above,is used to detect the degree or extent of binding. For instance, bylabeling the molecular beacon probe with a nonlinear-active label andmeasuring whether the label's orientation changes (via changes innonlinear optical intensity) at some interface, or in the bulk, one canstudy whether target strands are complementary, and the extend to whichthey are complementary since the amount of change that is measured innonlinear optical intensity can be correlated with the degree ofhybridization. In a specific embodiment, an Au nanoparticle is used toenhance the intensity of nonlinear optical radiation, such as secondharmonic generation scattered by an oxazole dye by several orders ofmagnitude when the nanoparticle and oxazole dye are in proximity to eachother. Upon hybridization of the probe to a complementary target, theintensity of the nonlinear optical radiation decreases and this decreasecan be quantitatively related to the amount of probe-targethybridization. The sensitivity of the technique is deternined by, amongother factors, the background nonlinear optical signal beforehybridization occurs.

[0497] Variations on Uses of the Invention

[0498] Although the present invention can be used in many scientificareas of analysis and in particular, in the chemical and biologicalarts, the present invention can be especially useful in drug discoveryor in fundamental studies where compounds (targets) are tested forbinding and and ability to activate probes, wherein the probes are ionchannel proteins, GPCR proteins, or other receptors, or other molecules.

[0499] A wide flexibility is provided for the apparatus. Scanning,imaging, detection techniques at a fixed position, etc. can all bereadily used with the present invention. Scanning of microarrays in theart includes confocal-based schemes and non-confocal based schemes. U.S.Pat. No. 5,834,758 (Trulson et al.—relevant portions of which areincorporated by reference herein) describes a non-confocal based schemefor imaging a microarray using fluorescence detection. However, thesample should lie very flat in order to image only within a single focalplane for good out-of-plane discrimination. Therefore, a very finelyadjustable translation stage requiring specialized components ispreferably be used for this purpose adding to the cost of the instrumentand possibly the lifetime as well. The image quality of this type ofapparatus can be sensitive to mechanical vibrations. Furthermore,discrimination of the out-of-plane (non-surface bound) fluorophoresplaces a limit on the sensitivity of the technique. U.S. Pat. No.6,134,002 (Stimson et al.—relevant portions of which are incorporated byreference herein) is an example of a confocal scanning microscope devicefor imaging a sample plane, i.e. a microarray. Although theconfocal-based techniques have good depth discrimination, the scan ratemay be low due to descanning requirements and the light throughput canbe low, reducing the overall signal to noise ratio and the sensitivityof the technique.

[0500] The invention can be used for studying binding processes betweenother biological components: cells with viruses; protein-proteininteractions; protein-ligand; cell-ligand; protein-drugs, nucleicacid-drugs, cell-small molecule; cell-nucleic acid; peptide-cell, oligoor polynucleotides, virus-cell, protein-small molecule, etc., and ingeneral, any binding reaction which results in a conformational change.Biomimetic membranes such as phospholipid supported bilayers (eg., eggphosphatidylcholine) can also be used and are particularly useful whenstudies involve membrane protein probes.

[0501] Probes, targets, receptors, etc. can be rendered nonlinear-active(made to possess a hyperpolarizability) by direct labeling or by using adecorator molecule or other candidate that has a binding affinity forthe probes or targets, and is itself intrinsically or renderednonlinear-active, and which will respond to a conformational change onthe probes or targets by shifting its position, by virtue of themolecular bond that binds the decorator to the probe or target. Anantibody to a receptor is an example of such a decorator molecule. Thedecorator molecule should not itself block the active region of thereceptor so that potential agonists, inhibitors, activators, etc. canbind to and produce action on the receptor.

[0502] Another example of the invention's use is to label receptors orother components that have an affinity for some virus. When the virusbinds or interacts with the receptor or other component, thisinteraction will affect the orientation of the label with respect to thedirection of the fundamental beam, and thus change the properties of themeasured nonlinear optical light (e.g., the intensity of the nonlinearlight).

[0503] Other examples of the technique's use with arrays includecellular arrays, supported lipid bilayer arrays with or without membraneor attached proteins, etc. Many methods exist in the art for couplingbiomolecules (eg., nucleic acid, protein and cells) to solid supports inarray format. A wide degree of flexibility may be used in providing themeans by which the arrays are created. They can involve, for example,covalent or non-covalent coupling to the substrate directly, to achemically derivatized substrate, to an intermediate layer of some kind(e.g., self-assembled monolayer, a hydrogel or other bio-compatiblelayer known in the art). The identity of the probes (e.g., proteinstructure or oligonucleotide sequence) can vary from site to site acrossthe solid surface, or the same probe can uniformly cover the surface.Targets can be of a single identity or a combination of targets withdifferent identities. The arrays can be prepared in a variety of waysincluding, but not limited to, ink-jet printing, photolithography,micro-contact printing, or any other manner known to one skilled in theart of fabricating them.

[0504] Because the binding process can be measured in real time and inthe presence of bulk biological components due to thesurface-selectivity of the nonlinear optical technique, equilibriumbinding curves and kinetics can be measured, the bulk concentration ofthe components can be varied, and a “wash-away” step to remove unboundcomponents, as is used with fluorescence-based detection, may beunnecessary.

[0505] A wide degree of flexibility is expected in the design of theapparatus including, but not limited to, the source of the fundamentallight, the optical train necessary to control, focus or direct thefundamental and nonlinear light beams, the design of the array, thedetection system, and the use of a grating or filters and collectionoptics. The mode of generation (irradiation) or collection can be variedincluding, for example, the use of evanescent wave (total internalreflection), planar wave guide, reflection, or transmission geometries,fiber-optic, near-field illumination, confocal techniques or the use ofa microcavity for power build-up, or integrating detection system suchas an integrating sphere. A number of methods for scanning a microarrayon a solid surface can be adapted for use. Examples include U.S. Pat.Nos. 5,834,758 to Trulson et al. (1998), 6,025,601 to Trulson et al.(2000), 5,631,734 Stem et al. (1997), and 6,084,991 to Sampas(2000)—relevant portions of which are incorporated by reference herein.

[0506] Because the second harmonic light beam makes a definite angle tothe surface plane, one can read-out the properties of the nonlinearoptical radiation, for instance, as a function of fundamental incidenceposition in a two-dimensional array format, without needing tomechanically translate the detector or sample and without extensivecollection optics. In the ‘beam scanning’ embodiment, a mechanicaltranslation of sample surface or detector is not required—only a changein a direction and/or angle of the fundamental incidence on the sample(for a fixed sample and detector)—the apparatus offers much fasterscanning capability, improved ease of manufacturing and a longerlifetime.

[0507] When using the present invention to study an interface, theinterface can comprise a silica, glass, silicon, silicon nitride,polystyrene, nylon, plastic, a metal, semiconductor or insulatorsurface, or any mixtures thereof, or any surface to which probes, suchas biological components, can adsorb or be attached. The interface canalso include biological cell and liposome surfaces. The attachment orimmobilization can occur through a variety of techniques well known inthe art. For example, oligonucleotides can be prepared via techniquesdescribed in “Microarray Biochip Technology”, M., Schena (Ed.), EatonPublishing, 1998—relevant portions of which are incorporated byreference herein. And, for example with proteins, the surface can bederivatized with aldehyde silanes for coupling to amines on surfaces ofbiomolecules (G. MacBeath and S. L. Schreiber, “Printing Proteins asMicroarrays for High-Throughput Function Determination”, Science 2000,289, 1760-1763—relevant portions of which are incorporated by referenceherein). BSA-NHS (BSA-N-hydroxysuccinimide) surfaces can also be used byfirst attaching a molecular layer of BSA to the surface and thenactivating it with N,N′-disuccinimidyl carbonate. The activated lysine,aspartate or glutamate residues on the BSA react with surface amines onthe proteins.

[0508] The present invention can be applied to an ensemble of moleculesor to a single molecule—i.e., ensemble reaction measurements or asingle-molecule reaction measurement.

[0509] Supported phospholipid bilayers can also be used, with or withoutmembrane proteins or other membrane-associated components as, forexample, in Salafsky et al., Architecture and function of membraneproteins in planar supported bilayers: A study with photosyntheticreaction centers'” Biochemistry 35 (47): 14773-14781 (1996)—relevantportions of which are incorporated by reference herein, “Biomembranes”,Gennis, Springer-Verlag, Kalb et al., 1992 and Brian et al., “AllogeneicStimulation of Cyto-toxic T-cells by Supported Planar Membranes,”PNAS—Biological Sciences 81 (19): 6159-6163 (1984)—relevant portions ofwhich are incorporated herein. Supported phospholipid bilayers are wellknown in the art and there are numerous techniques available for theirfabrication, with or without associated membrane proteins. Thesesupported bilayers typically should be submerged in aqueous solution toprevent their destruction when they become exposed to air.

[0510] Probes can be part of a biological cell, liposome, bead, etc.that naturally form an interface capable of generating nonlinear opticalradiation due to their size (although they are nominallycentrosymmetric, their diameter is of the order of the wavelength oflight in the visible spectrum and this allows for generation of thenonlinear optical light according to well known art). Alternatively,probes can be molecules or particles (e.g., detergent-solubilizedreceptors) that are induced to orient by the application of an electricfield. The use of electric fields to create a non-centrosymmetric regioncapable of generating nonlinear radiation—e.g., second harmonic, sumfrequency or difference frequency generation, is well known in the priorart. One aspect of this prior art is often called ‘EFISH’—electricfield-induced second harmonic generation. In a specific embodiment, anapplied electric field is used to pole molecules within a region tocreate an ordered (non-centrosymmetric) region within a phase ormaterial; the resulting region is then capable of generating nonlinearoptical radiation.

[0511] In a specific embodiment, the probe-target hybridization can bemeasured by detecting the intensity of nonlinear optical light (e.g.,second harmonic light) at some position on a substrate withsurface-attached probes; the intensity of the second harmonic lightchanges as labeled targets bind to the probes at the surface and becomepartially oriented because of the binding, thus satisfying thenon-centrosymmetric condition for generation of second harmonic light atthe interface. Modeling of the intensity of light with concentration ofprobe-target binding complexes at the interface can be accomplishedusing a variety of methods, for instance by calibrating the techniquefor a given probe-target interaction using radiolabels or fluorescencetags. Controls for non-specific binding of targets to the surface can beperformed according to procedures well known to one skilled in the art,for example: i) addition of deliberately non-complementary targets andmeasuring for surface-selective nonlinear optical signal, or ii) addingblockers which are known to prevent probe-target binding, addingcomplementary targets and measuring the resulting surface-selectivenonlinear optical signal. In case i) the surface-selective nonlinearoptical signal change upon addition of non-complementary targets will besubstantially lower than upon addition of complementary targets. In caseii) the signal change will be significantly lower in the presence ofblocker than in the absence of blocker.

6. EXAMPLES Example 6.1

[0512] A Molecular Beacon analogue (MB analogue) oligonucleotide,coupled to a nonlinear-active dye, and purified, is purchased from acommercial source such as Midland Certified Reagent Company (Midland,Tex.). The nonlinear-active oxazole dye used is oxazole (SE)1-(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium bromide (PyMPO, SE: Molecular Probes Corp.)attached via an amine group at the 3′ end.

[0513] The oligonucleotide is placed into the sample well of an EFISHcell. There are a variety of EFISH cells available in the art. Thesample cell described in the publication by C. G. Bethea (‘Experimentaltechnique of dc induced SHG in liquids: measurements of the nonlinearityof CH₂I₂”, Applied Optics 1975, 14, 1447) is used. The direction of theapplied electric field is parallel to the electric field of the laserbeam. A commercial femtosecond mode-locked system (Mira 900 and Verdi5W) is used as the fundamental source. The fundamental is directed intothe region of the cell between the electrodes. The back-reflected secondharmonic is blocked from entering the oscillator using a color filter;fundamental light beyond the sample is blocked using a color filter. Thesecond harmonic light is collected and focused onto a monochromator(CM110 CVI Laser) using plano-convex lenses (Melles Griot Inc.). Thelight is detected using a Hamamatsu photomultiplier tube with a Bertanpower supply. The signals from the photomultiplier are sent to aStanford Research Systems SR400 photon counting unit and processed usinga PC. An electric field is applied using a Bertan power supply andhome-built electronics to pulse and synchronize the field with the laserpulses.

[0514] The dye-labeled MB analogue probes are poled by application ofthe electric field when it is on. By comparing the average secondharmonic intensity of the poled MB analogues in the absence and presenceof target, the binding affinity (or sequence if it is unknown) of thetarget to the MB analogue can be measured. The SHG signal in the absenceof target represents a background measurement that is used as a‘baseline’ to compare with the signal in the presence of the target.

[0515] Upon addition of a perfect complementary target, the MB analogueprobe is activated and this leads to a conformational change andtherefore to a change in the average orientation of the nonlinear-activedye. Because the intensity of the measured SHG light is proportional tothe average orientation of the nonlinear-active dye, a change in dyeorientation results in a change in the intensity of the second harmonicbeam. Targets with less than perfect complementarity to the-MB analogueprobe sequence will bind with a lower affinity to the MB analogue probeand will therefore activate a lower proportion of the MB analogue probemolecules and cause a smaller amount of the conformational change. Arelationship between binding affinity and the intensity of the secondharmonic beam can be readily developed according to procedures known inthe art that relate a change in nonlinear intensity to concentration ofthe nonlinear-active species.

Example 6.2

[0516] The β2 adrenergic receptor, a GPCR protein, is purified anddetergent-solubilized according to well known procedures (e.g., Ghanouniet al., 98(11): 5997 PNAS). The protein is labeled at an endogenouscysteine (Cys-265) with1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridiniumtrifluoromethanesulfonate (PyMPO epoxide, Molecular Probes), anonlinear-active dye, at 1:1 stoichiometry following standard proceduresand using the work of Ghanouni et al., 98(11): 5997 PNAS, as a guide.The nonlinear-active dye is attached to a part of the protein thatundergoes a conformational change when the protein is activated. Afterseparation of the non-covalently bound dye, the receptor is placed in amedium situated between two electrodes and through which passes afundamental beam (e.g., output of ˜1W avg. Power, ˜150 fs pulses from aTi:Sapphire system such as the Verdi-Mira commercial system fromCoherent Inc.). The apparatus and sample preparation (e.g., applicationof electric field, electric field induced second harmonicgeneration—EFISH) are well known to one of ordinary skill in theart—standard optics are used to focus the fundamental on the sample andto collect the second harmonic radiation. FIG. 18 depicts the apparatusset-up and FIG. 17 depicts the sample holder with applied electricfield. An electric field is applied across the medium according to FIG.17, partially aligning the receptor and its bound dye and creating anon-centrosymmetric condition necessary for observation of SHG. Abackground signal intensity is measured at 400 nm using a color filter(BG-39, CVI Laser) to block the fundamental, a monochromator to selectwavelength (CM110, CVI Laser), a PMT (Hamamatsu) and single-photoncounting electronics (SR-400, Stanford Research Systems). The wavelength400 nm is selected to create a resonance enhancement effect via anelectronic transition of the dye. Ligand is added to the medium—a drugcandidate that is tested for binding, for example; if the ligand (drug)binds to the receptor, a conformational change in the probe (thereceptor) is induced which changes the nonlinear optical signalintensity. Known ligands (isoproterenol) and partial agonists(epinephrine, salbutamol and dobutamine) are used to calibrate thenonlinear optical response with amount of conformational change, i.e.binding affinity.

[0517] In an alternate embodiment, the labeling reaction can be carriedout using various coupling chemistries and/or stoichiometries(label:probe) to determine which coupling chemistry gives the optimalsignal in the nonlinear optical measurement. For instance, it may not beknown a priori which sub-parts of the probes actually undergo aconformational change (positional shift) due to target activation and byundertaking a variety of labeling reactions (known in the art to benecessary to find optimal labeling conditions in fluorescence labeling,etc.), it can be determined which chemistries lead to a label thatundergoes conformational change when the probe is activated.

[0518] In an alternative embodiment, ion channels or receptors such asGPCR receptors are labeled directly in biological cells withnonlinear-active labels and/or enhancers. For instance, the publicationby Glauner et al. (Nature, v.402 813 (1999)) demonstrates fluorescentlabeling of a Shaker potassium ion channel in whole cells. A backgroundsignal is measured. When agonist binds to the receptors, it induces aconformational change in the receptors, changing the orientation of thelabels and thus the nonlinear optical signal (e.g., the signalintensity). A surface-selective nonlinear optical apparatus is used thusto measure ligand-gated conformational changes in the receptors via thelabels and/or enhancers in whole cells. Fundamental light is focusedonto a cell layer that has been cultured on Becton-Dickinson Falconplates, for instance—and the second or higher harmonic is collectedaccording to procedures well known in the art. In an alternativeembodiment, the labeled cells are suspended in a medium and fundamentallight is focused into a region of the sample containing these cells.FIG. 18 depicts an embodiment of this apparatus. Second harmonic lightis collected according to procedures known in the art, for instance at 0or 90 degrees with respect to the fundamental beam.

[0519] In an alternative embodiment, enhancers are suspended ordissolved in the medium with labeled cells or molecules to enhance thenonlinear response of the cells or molecules.

[0520] In an alternative embodiment, probes are placed in artificialmembranes or liposomes. If probes are expressed in cells, then can bepurified and reconstituted into these membranes according to procedureswell known in the art.

[0521] In an alternative embodiment, the probes are in contact with,cultured on or patterned on surface that is itself in contact with aprism (or is the underside of the prism). The prism allows totalinternal reflection of the fundamental at the interface containing theprobes and thus high Fresnel factors (electric field amplitudes) leadingto higher nonlinear optical signals. In this mode of the set-up, thefundamental beam undergoes total internal reflection at the interfacecontaining the probes and its evanescent wave is used to generate thenonlinear light. FIG. 2 illustrates an embodiment of this type. In FIG.2, an index matching material or liquid (75) is used to couple the prism(70) to a substrate containing the microarray (80) in contact withsolution containing targets (85), whereby total internal reflectionoccurs at the interface between material (80) and solution (85). Theprism material can be, for example, BK7 type glass (Melles Griot) andthe index matching material obtained commercially from Corning Corp. orNye Corp.

[0522] In an alternative embodiment, the experimental set-up is asdescribed in Salafsky and Eisenthal, “Protein adsorption at interfacesdetected by second harmonic generation,” J. Phys. Chem. B, 104 (32):7752-7755 (2000), Salafsky and Eisenthal, “Second harmonic spectroscopy:detection and orientation of molecules at a biomembrane interface,”Chem. Phys.Lett. 319 (5-6): 435-439 (2000), and references set forththerein. A femtosecond pulsed laser (Mail-Tai, Spectra-Physics) is usedas the source of fundamental light at 800 nm operating at 80 MHz with<200 fs pulses at 1 W average power. The laser beam directed onto theentrance aperture of a Dove prism (Melles Griot, BK-7) and focused witha concave lens (Oriel) (spot size ˜50 micron diameter). The Dove prismis mounted in a teflon holder and in contact with buffer or distilledwater. The beam undergoes total internal reflection (evanescent wavegeneration) within the prism and the fundamental and second harmonicbeams emerge roughly collinearly from the exit aperture. A color filteris used to block the fundamental light while passing the second harmonicto a monochromator (2 nm bandwidth slit). The monochromator is scannedfrom 380-500 nm to detect the second harmonic spectrum. If necessary,the fundamental light wavelength can be tuned as well. A single photoncounting detector and photomultiplier tube are used to detect the outputof the monochromator and a PC with software are used to record the dataand control the monochromator wavelength. A background second harmonicsignal is measured before addition of ligand or other stimulus toproduce or test for conformational change in the probes.

Example 6.3

[0523] Oligodeoxyribonucleotides with suitable structures for molecularbeacons are selected and synthesized according to procedures known toone of ordinary skill in the art with a primary amine at the 3′ end anda disulfide group at the 5′ end and a biotin group that replaces a dT.The following MB analogue can be used, for example: 5′-CCT AGC TCT AAATCG CTA TGG TCG CGC(Biotin dT)AG G-3′ (SEQ ID NO: 6). The amine-reactivenonlinear-active oxazole dye: oxazole (SE)1-(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium bromide (PyMPO, SE: Molecular Probes Corp.) isconjugated to the primary amine. In this coupling reaction, a 100 μlsolution containing 100 μM oligonucleotide dissolved in 0.1 M sodiumbicarbonate is reacted with 0.1 mg of the succinimidyl ester of the dyedissolved in 100 μl of dimethyl sulfoxide. The reaction mixture isstirred at room temperature for 2 hours. The reaction product ispurified with a Sephadex column (NAP-5; Amersham Pharmacia Biotech)equilibrated with 10 ml of 0.1 M triethylammonium acetate (pH 6.5).After purification, the 5′-end disulfide is cleaved and the freesulfhydryl is covalently attached to a 1.4 nm diameter gold cluster thatserves to enhance the nonlinear response of the oxazole dye (Nanogold;Nanoprobes), and which comes with one N-propylmaleimide and has beenpassivated with water-soluble phosphine ligands. The coupling of the Auparticle is achieved according to procedures well known to one ofordinary skill in the art. For example: the disulfide bond is cleavedwith dithiothreitol (DTT), and the oligonucleotide is purified of excessDTT before coupling to the gold. An amount of 10 μl of 1 M DTT is addedto 25 μl of oligonucleotide mixed with 75 μl of sodium bicarbonate, pH8.3. After a one hour incubation, the oligonucleotide solution ispurified using reverse-phase chromatography, as described. The fractionscontaining the activated oligonucleotide are purified using a Sephadexcolumn (NAP-5) equilibrated with water. Part of the elution product (37pmol to 370 pmol of DNA, suspended in 180 μl of water) is immediatelyreacted with 6 nmol of the monomaleimido—gold particles (Nanoprobes) inaqueous 20 mM NaH2PO4, 150 mM NaCl, 1 mM ethylenediamine tetraethylacetate (EDTA) buffer, pH 6.5, containing 10% isopropanol at 4° C. for24 h. Reaction products are analyzed by gel electrophoresis on a 10%nondenaturing acrylamide gel performed in Tris-borate EDTA (TBE) at 10V/cm.

[0524] The biotinylated MB analogue with attached nonlinear-active dyeand gold nanoparticles are coupled to streptavidin-derivatized glassaccording to well known procedures. For example, the biotinylated MBanalogues are immobilized on the etched portion of a glass fiber. Abatch of optical fibers is used in a single immobilization cycle. About2 cm of cladding is stripped away from the core by chemical etching atone end of the fiber probe. The fiber probe is perpendicularly dippedinto a 49% hydrofluoric acid solution for 12 min. The HF solution iscovered by heptane solvent. The etched fiber probe is washed withultrapure water before being used for the subsequent immobilizationexperiment.

[0525] Biotinylated MB analogues are immobilized on the etched portionof the fiber for DNA sensing. The etched fiber probes are first cleanedby immersion in a 1:1 v/v concentrated HCl/MeOH mixture for 30 min.,rinsed in water, and submerged in concentrated sulfuric acid for 30 min.Further rinsing and then boiling in water for 8-10 min follows.Silanization of the fibers is performed by immersing them in a freshlyprepared 1% (v/v) solution of DETA(Trimethoxysilylpropyldiethylenetriamine purchased from United ChemicalTechnologies, Bristol Pa.) in 1 mM acetic acid for 20 min at roomtemperature. The DETA-modified fiber probes are thoroughly rinsed withwater to remove excess DETA. The silanized fiber probes are dried undernitrogen and fixed by heating in a 120° C. oven for 5 min. Then thesilanized fibers are immersed in 0.5 mg/ml NHS-LC-biotin(sulfosuccinimidyl-6-(biotinamido) hexanoate purchased from Pierce,Rockford IL) in 0.1 M bicarbonate buffer (pH 8.5) for 3 hours at roomtemperature. Streptavidin (Sigma, St. Louis Mo.) is bound to the fibersurface by incubating the biotinylated fibers overnight at 4° C. in asolution containing 1.0 mg/ml of the streptavidin. Thestreptavidin-immobilized optical fibers are then immersed with abiotinylated MB analogue solution (10⁻⁶ M in 10 mM phosphate buffer atpH 7.0) for as long as 20 min or overnight at 4° C. to allow thebiotinylated MB analogue to be immobilized on the surface.

[0526] The optical fibers are interrogated optically using secondharmonic generation by propagating a fundamental beam down the fiber andmeasuring the intensity of the second harmonic beam back-reflected afterit interacts evanescently with the MB analogue at the distal end of thefiber. In the absence of complementary targets, the oligonucleotideproduces a large amount of second harmonic light due to the proximity ofthe oxazole dye and the Au particle in the hairpin-loop structure of theMB analogue; the intensity of the second harmonic light in this case canserve as a background. In the presence of complementary targets, thehybridization reaction causes the dye and the Au particle to spatiallyseparate, greatly reducing the intensity of the second harmonic lightdetected. A linear relationship can be constructed between the amount ofhybridization that occurs and the intensity of the second harmonic lightand thus the derivatized optical fibers with detection system serves asan optical device for detection of complementary targets to the selectedprobe.

Example 6.4

[0527] Glass microspheres which are optically encoded with fluorescentdyes are derivatized with the MB analogues and an array of microsphereswith distinct oligonucleotide probes is prepared at the distal end of anoptical fiber as found in the art (e.g., U.S. Pat. No. 5,250,264, Waltet al.; U.S. Pat. No. 5,298,741 Walt et al.; U.S. Pat. No. 5,252,494Walt et al; U.S. Pat. No. 6,023,540 Walt et al.; U.S. Pat. No. 5,814,524Walt et al.; U.S. Pat. No. 5,244,813 Walt et al.; U.S. Pat. No.5,512,490 Walt et al. and commercially (Illumina Corporation, forexample). These arrays can be used to detect multiple target sequences.Alternatively, the optical encoding can be accomplished usingnonlinear-active dyes with different spectral characteristics from thebeacon-associated nonlinear-active dye so that the both the encoding andthe hybridization detection- can be made using a nonlinear opticaltechnique such as second harmonic generation.

[0528] If the nonlinear MB analogues are used in homogeneous solution,an applied electric field can be applied to the MB analogues in an EFISHmethod to create the required non-centrosymmetric condition.

[0529] In an alternative embodiment, one is interested in finding drugs,antagonists, agonists or other species which block or reduce the bindingof the MB analogues with targets—these compounds may be referred to as‘inhibitors’ or ‘blockers.’ In this application, labeled targets arebound to probes at the interface. The inhibitors are added to thesample, and if the particular species being tested is successful inblocking or reducing the probe-target binding, the nonlinear opticallight measured will change—the background radiation in this embodimentis due to target-probe binding; the displacement of the targets from theprobes at the interface by the inhibitors leads to a change in thenonlinear optical light measured, for instance as a decrease inintensity of the nonlinear radiation generated by the interface or awavelength shift in the nonlinear radiation spectrum.

[0530] In an optional embodiment, controls to determine degree ofnon-specific nonlinear optical signals (e.g., not due to specificprobe-target binding) can be performed according to standard procedureswell known to one skilled in the art. In nucleic acid microarrays, forexample, the intensity of the nonlinear optical signal at regionsbetween the probe-containing regions will produce a background signalthat can increase somewhat (but is substantially smaller than the signaldue to specific probe-target binding reactions) upon addition of targetsthat are either complementary or non-complementary to the probes. Thisbackground signal can be accounted for by, for example, adding onlynon-complementary probes and measuring the nonlinear optical signal inregions containing probes and not containing probes. Measuring thenonlinear optical signals in the presence of blockers known to preventprobe-target binding is another control technique well known to oneskilled in the art for determining the amount of background orartifactual signal present in a larger signal of interest, in this casethe specific probe-target binding reaction.

[0531] In another embodiment of the invention, the amine-reactiveoxazole dye (SE) 1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl) oxazol-2-yl)pyridinium bromide (PyMPO,SE: Molecular Probes Corp.) is reacted with a 1:1 molar ratio ofethylenediamine under the conditions specified by the Molecular Probesdirection and is allowed to react to completion. The oxazole-based dyenow contains a single amine group. This can be coupled to the primaryamine on an oligonucleotide using a homobifunctional crosslinking agent(Pierce, Rockford Ill.).

[0532] In an alternative embodiment, a-nonlinear-active-dye withattached biotin can be synthesized according to procedures known to oneof ordinary skill in the art to create dye-biotin molecules that are thenonlinear-active analogues of the biotin-fluorescent dye moleculesuseful for immobilization of oligonucleotides.

Example 6.5

[0533] A peptide-nucleic analogue (PNA) molecular beacon (hairpin-loopstructure) of 18-25 base-pairs sequence is labeled with PyMPO, SE(Molecular Probes Corp.) at the 3′ or 5′ end (or, alternatively, atcytosine base-pair locations) according to procedures well establishedin the art. The PNA is placed in purified water solution and orientedwith an applied electric field to generate a non-centrosymmetric region.Addition of PNA molecules with complementary sequence to the labeled PNAwill cause a conformational change and thus a change in measuredproperties of the nonlinear optical beams.

[0534] PNAs (linear, non-hairpin loop) can also be attached to glass orsilica surfaces according to procedures well known to those skilled inthe art. Addition of sequence-complementary DNA to purified water incontact with the glass surface containing the PNAs results in aconformational change in the PNAs on the surface, and thus a change inthe measured physical properties of the nonlinear optical light beam.

7. MISCELLANEOUS

[0535] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

[0536] All references cited herein are incorporated herein by referencein their entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

[0537] The citation of any publication is for its disclosure prior tothe filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

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1 6 1 20 DNA Artificial Sequence Description of Aritificial SequenceOligonucleotide structure for molecular beacon 1 aaaaaaaaaa aaaaactcgc20 2 16 DNA Artificial Sequence Description of Aritificial SequenceOligonucleotide structure for molecular beacon 2 gaaaaaaaaa aaaaaa 16 316 DNA Artificial Sequence Description of Aritificial SequenceOligonucleotide structure for molecular beacon 3 gaaaaaaaca aaaaaa 16 469 DNA Artificial Sequence Description of Aritificial SequenceOligonucleotide structure for molecular beacon 4 ctacctacag taccagcttnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnt tactcgaggg 60 atcctagtc 69 5 25 DNAArtificial Sequence Description of Aritificial Sequence Oligonucleotidestructure for molecular beacon 5 gcgactttt tttttttttt tctcgc 25 6 31 DNAArtificial Sequence Description of Aritificial Sequence Oligonucleotidestructure for molecular beacon 6 cctagctcta aatcgctatg gtcgcgctag g 31

1. A method for screening one or more candidate binding partners forbinding to a test molecule comprising: (a) illuminating a sample withone or more light beams at one or more fundamental frequencies, saidsample comprising said test molecule exposed to said one or morecandidate binding partners; and (b) measuring one or more physicalproperties of a nonlinear optical light beam emanating from said sample;wherein a change in the value of said one or more physical propertiesmeasured in step (b) relative to a value for said one or more physicalproperties measured in the absence of exposure of said test molecule tosaid one or more candidate binding partners indicates that said one ormore candidate binding partners bind said test molecule.
 2. The methodof claim 1, wherein said test molecule is bound to one or morenonlinear-active labels.
 3. The method of claim 2 further comprising thestep of binding said labels to said test molecule prior to step (a). 4.The method of claim 2, wherein said one or more labels are covalentlyattached.
 5. The method of claim 2, wherein said test molecule isnon-covalently bound to a molecule that is nonlinear-active.
 6. Themethod of claim 2, wherein said test molecule and one of saidnonlinear-active labels form a fusion protein.
 7. The method of claim 2,wherein said one or more labels comprise green fluorescent protein orderivatives or mutants of said protein that are nonlinear-active.
 8. Themethod of claim 2, wherein said labels are caged.
 9. The method of claim2, wherein said labels are molecular beacon analogues.
 10. The method ofclaim 2, wherein ultraviolet light acts to cleave a bond between saidnonlinear-active label and said test molecule.
 11. The method of claim2, wherein the nonlinear-active properties of said labels can be changedby exposure to a chemical agent or one or more light beam.
 12. Themethod of claim 1, wherein said candidate binding partners are bound toone or more nonlinear-active labels.
 13. The method of claim 12 furthercomprising the step of binding said labels to said candidate bindingpartners prior to step (a).
 14. The method of claim 12, wherein said oneor more labels are covalently attached.
 15. The method of claim 12,wherein said candidate binding partners are non-covalently bound to amolecule that is nonlinear-active.
 16. The method of claim 12, whereinsaid candidate binding partner and one of said nonlinear-active labelsform a fusion protein.
 17. The method of claim 12, wherein said one ormore labels comprise green fluorescent protein or derivatives or mutantsof said protein that are nonlinear-active.
 18. The method of claim 12,wherein said labels are caged.
 19. The method of claim 12, wherein saidlabels are molecular beacon analogues.
 20. The method of claim 12,wherein ultraviolet light acts to cleave a bond between saidnonlinear-active label and said candidate binding partners.
 21. Themethod of claim 12, wherein the nonlinear-active properties of saidlabels can be changed by exposure to a chemical agent or one or morelight beam.
 22. The method of claim 1, wherein the one or more physicalproperties are intensities.
 23. The method of claim 1, wherein the oneor more physical properties are polarization directions.
 24. The methodof claim 1, wherein said test molecule is nonlinear-active in theabsence of an exogenous nonlinear-active label bound to the testmolecule.
 25. The method of claim 1, wherein said one or more lightbeams have a wavelength in the range of 10 to 10000 nanometers.
 26. Themethod of claim 1, which further comprises comparing the value of saidphysical properties measured in step (b) with the value of the physicalproperties measured in the absence of exposure to said one or morecandidate binding partners.
 27. The method of claim 1, wherein said testmolecule or said candidate binding partners are bound to at least twodistinguishable nonlinear-active labels, wherein said one or more lightbeams are multiple light beams, and step (b) comprises measuring one ormore physical properties of at least two nonlinear optical light beamsemanating from said sample.
 28. The method of claim 1, wherein said testmolecule is purified.
 29. The method of claim 1, wherein said testmolecule is a G protein-coupled receptor (GPCR).
 30. The method of claim29, wherein said GPCR is bound to a nonlinear-active label.
 31. Themethod of claim 1, wherein said test molecule is a labeled GPCR.
 32. Themethod of any claims 1, wherein said nonlinear optical light beam issecond harmonic generated light.
 33. The method of claim 1, whichfurther comprises applying an electric field to said sample.
 34. Themethod of claim 33, wherein the electric field is stationary in time,time-varying, or some combination thereof.
 35. The method of claim 1,wherein said sample further comprises a bulk phase.
 36. The method ofclaim 35, wherein an electric field is applied to said bulk phase. 37.The method of claim 36, wherein the electric field is stationary intime, time-varying, or some combination thereof.
 38. The method of claim1, wherein said one or more nonlinear optical light beams are secondharmonics, third harmonics, sum frequencies or difference frequencies ofthe one or more fundamental frequencies.
 39. The method of claim 1,wherein said test molecule is part of a cell, liposome or membranesurface.
 40. The method of claim 1, which further comprises measuringsaid one or more physical properties of the nonlinear optical light beamin the absence of either the test molecules or the candidate bindingpartners in said sample.
 41. The method of claim 1, wherein said testmolecule is part of a natural or artificial membrane.
 42. The method ofclaim 1, wherein said one or more candidate binding partners are part ofa supported membrane, liposome or a biological cell.
 43. The method ofclaim 1, wherein said one or more candidate binding partners are coupledor conjugated in vitro to a solid surface.
 44. The method according toclaim 43, wherein said solid surface supports a phospholipid orartificial bilayer membrane.
 45. The method according to claim 44,wherein said phospholipid or artificial bilayer comprises membraneproteins.
 46. The method of claim 1, wherein said one or more candidatebinding partners comprise a portion of a surface of biological cells,liposomes, vesicles, beads, metal particles, or non-metal particles. 47.The method of claim 1, wherein said one or more candidate bindingpartners are patterned on a solid surface.
 48. The method of claim 47,wherein said oligonucleotides or polynucleotides are attached to regionson the surface of size nanometers to microns in dimension.
 49. Themethod of claim 1, wherein said one or more candidate binding partnersare patterned in an array format on a solid surface.
 50. The method ofclaim 49, wherein said one or more candidate binding partners comprise aplurality of different oligonucleotides or polynucleotides, saiddifferent oligonucleotides or polynucleotides each comprising adifferent sequence and attached to a different region on a solidsurface.
 51. The method of claim 49, wherein the oligonucleotides orpolynucleotides are patterned in a microarray format.
 52. The method ofclaim 1, wherein said one or more candidate binding partners comprise aplurality of different proteins, said different proteins each comprisinga different amino acid sequence and attached to a different region on asolid surface.
 53. The method of claim 52, wherein said one or morecandidate binding partners comprising a different amino acid sequenceare each attached to a different region on a surface of size 1 to 1000nanometers.
 54. The method of claim 52, wherein said proteins areattached to regions on the surface of size 1 to 1000 microns.
 55. Themethod of claim 1, wherein said one or more candidate binding partnerscomprise proteins patterned in an array format.
 56. The method of claim1, wherein the step of illuminating involves reflection, transmission,evanescent wave, multiple internal reflection, near-field optics,confocal, optical cavity, planar waveguide, fiber-optic ordielectric-slab waveguide.
 57. The method of claim 1, wherein the stepof measuring involves reflection, transmission, evanescent wave,multiple internal reflection, near-field optics, confocal, opticalcavity, planar waveguide, fiber-optic or dielectric-slab waveguide. 58.The method of claim 1, wherein said one or more candidate bindingpartners are molecular beacon analogues.
 59. The method of claim 1,wherein said one or more candidate binding partners are attached to asurface.
 60. The method of claim 59, wherein said surface is a metalsurface, a semiconductor surface, a glass surface, a latex surface, agel substrate, a fiber-optic surface, a silica surface or a beadsurface.
 61. The method of claim 59, wherein said surface is anon-planar surface.
 62. The method of claim 59, wherein said surface ischemically derivatized.
 63. The method of claim 62, wherein said surfaceis derivatized with a self-assembled monolayer.
 64. The method of claim62, wherein said surface is derivatized with an organosilane.
 65. Themethod of claim 1, wherein the step of measuring is repeated overdifferent periods of time.
 66. The method of claim 1, wherein said oneor more candidate binding partners is attached to a self-assembledmonolayer.
 67. The method of claim 66, wherein the self-assembledmonolayer is in the chemical family of silanes or terminal-functionalsilanes.
 68. The method of claim 1, wherein the one or more physicalproperties of the nonlinear optical light beam measured indicatethermodynamic or kinetic properties of said binding.
 69. The method ofclaim 1, wherein said binding involves a chemical bond, an electrostaticforce, physisorption, chemical affinity, chemisorption, molecularrecognition, physico-chemical binding, hydrogen bond or hybridizationprocess.
 70. The method of claim 1, wherein the nonlinear optical lightbeam is circularly polarized.
 71. The method of claim 1, wherein thesample is on an interface comprising a cell, liposome, vesicle surface,or a solid surface.
 72. The method of claim 1, wherein the samplefurther comprises one or more substances selected from the groupconsisting of decorator molecules, decorator particles, enhancers,modulators, inhibitors, molecular beacon analogues, and indicators. 73.The method of claim 1, wherein the mode of generation, collection ordetection of the nonlinear optical light beam is one or more modesselected from the group consisting of reflection, transmission,evanescent wave, multiple internal reflection, near-field opticaltechniques, confocal, optical cavity, planar waveguide, fiber-optic anddielectric-slab waveguide, and near-field techniques.
 74. The method ofclaim 1, wherein said test molecule is a drug or blocking agent.
 75. Amethod for screening one or more candidate modulator molecules for theability to modulate an interaction between a test molecule and itsbinding partner comprising: (a) illuminating a sample with one or morelight beams at one or more fundamental frequencies, said samplecomprising said test molecule exposed to (i) said binding partner, and(ii) said one or more candidate modulator molecules; and (b) measuringone or more physical properties of a nonlinear optical light beamemanating from said sample; wherein a change in the value of said one ormore physical properties measured in step (b) relative to the value forsaid one or more physical properties measured in the absence of exposureto said one or more candidate modulator molecules indicates that saidone or more candidate modulator molecules modulate the interactionbetween said test molecule and its binding partner.
 76. The method ofclaim 75, wherein the one or more physical properties are intensities.77. The method of claim 75, wherein the one or more physical propertiesare polarization directions.
 78. The method of claim 75, wherein saidtest molecule is nonlinear-active in the absence of an exogenousnonlinear-active label bound to the test molecule.
 79. The method ofclaim 75, wherein said one or more light beams have a wavelength in therange of 10 to 10000 nanometers.
 80. The method of any claims 75,wherein said nonlinear optical light beam is second harmonic generatedlight.
 81. The method of claim 75, wherein the step of illuminatinginvolves reflection, transmission, evanescent wave, multiple internalreflection, near-field optics, confocal, optical cavity, planarwaveguide, fiber-optic or dielectric-slab waveguide.
 82. The method ofclaim 75, wherein the step of measuring involves reflection,transmission, evanescent wave, multiple internal reflection, near-fieldoptics, confocal, optical cavity, planar waveguide, fiber-optic ordielectric-slab waveguide.
 83. The method of claim 75, wherein the stepof measuring is repeated over different periods of time.
 84. The methodof claim 75, wherein the one or more physical properties of thenonlinear optical light beam measured indicate thermodynamic or kineticproperties of said binding.
 85. The method of claim 75, wherein saidbinding involves a chemical bond, an electrostatic force, physisorption,chemical affinity, chemisorption, molecular recognition,physico-chemical binding, hydrogen bond or hybridization process. 86.The method of claim 75, wherein the sample further comprises one or moresubstances selected from the group consisting of decorator molecules,decorator particles, enhancers, modulators, inhibitors, molecular beaconanalogues, and indicators.
 87. The method of claim 75, wherein the modeof generation, collection or detection of the nonlinear optical lightbeam is one or more modes selected from the group consisting ofreflection, transmission, evanescent wave, multiple internal reflection,near-field optical techniques, confocal, optical cavity, planarwaveguide, fiber-optic and dielectric-slab waveguide, and near-fieldtechniques.
 88. The method of claim 75, wherein said test molecule ispurified.
 89. The method of claim 75, wherein said test molecule is a Gprotein-coupled receptor (GPCR).
 90. The method of claim 75, whereinsaid GPCR is bound to a nonlinear-active label.
 91. The method of claim75, wherein said test molecule is a labeled GPCR.
 92. The method ofclaim 75, which further comprises applying an electric field to saidsample.
 93. The method of claim 92, wherein the electric field isstationary in time, time-varying, or some combination thereof.
 94. Themethod of claim 75, wherein said sample further comprises a bulk phase.95. The method of claim 94, wherein an electric field is applied to saidbulk phase.
 96. The method of claim 95, wherein the electric field isstationary in time, time-varying, or some combination thereof.
 97. Themethod of claim 75, wherein said one or more nonlinear optical lightbeams are second harmonics, third harmonics, sum frequencies ordifference frequencies of the one or more fundamental frequencies. 98.The method of claim 75, wherein said sample further comprises one ormore candidate binding partners.
 99. The method of claim 98, whichfurther comprises comparing the value of said physical propertiesmeasured in step (b) with the value of the physical properties measuredin the absence of exposure to said one or more candidate bindingpartners.
 100. The method of claim 98, wherein said test molecule orsaid candidate binding partners are bound to at least twodistinguishable nonlinear-active labels, wherein said one or more lightbeams are multiple light beams, and step (b) comprises measuring one ormore physical properties of at least two nonlinear optical light beamsemanating from said sample.
 101. The method of claim 98, wherein saidone or more candidate binding partners are patterned in an array formaton a solid surface.
 102. The method of claim 101, wherein said one ormore candidate binding partners comprise a plurality of differentoligonucleotides or polynucleotides, said different oligonucleotides orpolynucleotides each comprising a different sequence and attached to adifferent region on a solid surface.
 103. The method of claim 101,wherein the oligonucleotides or polynucleotides are patterned in amicroarray format.
 104. The method of claim 98, wherein said one or morecandidate binding partners comprise proteins patterned in an arrayformat.
 105. The method of claim 98, wherein said one or more candidatebinding partners are molecular beacon analogues.
 106. The method ofclaim 98, wherein said one or more candidate binding partners areattached to a surface.
 107. The method of claim 106, wherein saidsurface is a metal surface, a semiconductor surface, a glass surface, alatex surface, a gel substrate, a fiber-optic surface, a silica surfaceor a bead surface.
 108. The method of claim 106, wherein said surface isa non-planar surface.
 109. The method of claim 106, wherein said surfaceis chemically derivatized.
 110. The method of claim 109, wherein saidsurface is derivatized with a self-assembled monolayer.
 111. The methodof claim 109, wherein said surface is derivatized with an organosilane.112. A method for detecting a conformational change in a test moleculeupon binding of the test molecule to a binding partner comprising:contacting said test molecule with one or more candidate bindingpartners, wherein the test molecule or the one or more candidate bindingpartners is labeled with a nonlinear-active moiety that is not native tothe test molecule or the one or more candidate binding partners,respectively; (a) illuminating said contacted test molecule with one ormore light beams at one or more fundamental frequencies; and (b)measuring one or more physical properties of a nonlinear optical lightbeam emanating from said sample; wherein a change in the value of saidone or more physical properties measured in step (b) relative to thevalue for said one or more physical properties measured in the absenceof said one or more candidate binding partners indicates that at leastone of said one or more candidate binding partners bind to said testmolecule and that said binding induces a conformational change in saidcandidate binding partners, in said test molecule, or in both saidcandidate binding partners and said test molecule.
 113. The method ofclaim 112, wherein the one or more physical properties are intensities.114. The method of claim 112, wherein the one or more physicalproperties are polarization directions.
 115. The method of claim 112,wherein said test molecule is nonlinear-active in the absence of anexogenous nonlinear-active label bound to the test molecule.
 116. Themethod of claim 112, wherein said one or more light beams have awavelength in the range of 10 to 10000 nanometers.
 117. The method ofclaim 112, which further comprises comparing the value of said physicalproperties measured in step (b) with the value of the physicalproperties measured in the absence of exposure to said one or morecandidate binding partners.
 118. The method of claim 112, wherein saidtest molecule or said candidate binding partners are bound to at leasttwo distinguishable nonlinear-active labels, wherein said one or morelight beams are multiple light beams, and step (b) comprises measuringone or more physical properties of at least two nonlinear optical lightbeams emanating from said sample.
 119. The method of claim 112, whereinsaid test molecule is purified.
 120. The method of claim 112, whereinsaid test molecule is a G protein-coupled receptor (GPCR).
 121. Themethod of claim 120, wherein said GPCR is bound to a nonlinear-activelabel.
 122. The method of any claims 112, wherein said nonlinear opticallight beam is second harmonic generated light.
 123. The method of claim112, which further comprises applying an electric field to said sample.124. The method of claim 123, wherein the electric field is stationaryin time, time-varying, or some combination thereof.
 125. The method ofclaim 112, wherein said sample further comprises a bulk phase.
 126. Themethod of claim 125, wherein an electric field is applied to said bulkphase.
 127. The method of claim 126, wherein the electric field isstationary in time, time-varying, or some combination thereof.
 128. Themethod of claim 112, wherein said one or more nonlinear optical lightbeams are second harmonics, third harmonics, sum frequencies ordifference frequencies of the one or more fundamental frequencies. 129.The method of claim 112, wherein said one or more candidate bindingpartners are patterned in an array format on a solid surface.
 130. Themethod of claim 129, wherein said one or more candidate binding partnerscomprise a plurality of different oligonucleotides or polynucleotides,said different oligonucleotides or polynucleotides each comprising adifferent sequence and attached to a different region on a solidsurface.
 131. The method of claim 129, wherein the oligonucleotides orpolynucleotides are patterned in a microarray format.
 132. The method ofclaim 112, wherein said one or more candidate binding partners compriseproteins patterned in an array format.
 133. The method of claim 112,wherein the step of illuminating involves reflection, transmission,evanescent wave, multiple internal reflection, near-field optics,confocal, optical cavity, planar waveguide, fiber-optic ordielectric-slab waveguide.
 134. The method of claim 112, wherein thestep of measuring involves reflection, transmission, evanescent wave,multiple internal reflection, near-field optics, confocal, opticalcavity, planar waveguide, fiber-optic or dielectric-slab waveguide. 135.The method of claim 112, wherein said one or more candidate bindingpartners are molecular beacon analogues.
 136. The method of claim 112,wherein said one or more candidate binding partners are attached to asurface.
 137. The method of claim 136, wherein said surface is a metalsurface, a semiconductor surface, a glass surface, a latex surface, agel substrate, a fiber-optic surface, a silica surface or a beadsurface.
 138. The method of claim 136, wherein said surface is anon-planar surface.
 139. The method of claim 136, wherein said surfaceis chemically derivatized.
 140. The method of claim 139, wherein saidsurface is derivatized with a self-assembled monolayer.
 141. The methodof claim 139, wherein said surface is derivatized with an organosilane.142. The method of claim 112, wherein the step of measuring is repeatedover different periods of time.
 143. The method of claim 112, whereinsaid one or more candidate binding partners is attached to aself-assembled monolayer.
 144. The method of claim 143, wherein theself-assembled monolayer is in the chemical family of silanes orterminal-functional silanes.
 145. The method of claim 112, wherein theone or more physical properties of the nonlinear optical light beammeasured indicate thermodynamic or kinetic properties of said binding.146. The method of claim 112, wherein said binding involves a chemicalbond, an electrostatic force, physisorption, chemical affinity,chemisorption, molecular recognition, physico-chemical binding, hydrogenbond or hybridization process.
 147. The method of claim 112, wherein thesample further comprises one or more substances selected from the groupconsisting of decorator molecules, decorator particles, enhancers,modulators, inhibitors, molecular beacon analogues, and indicators. 148.The method of claim 112, wherein the mode of generation, collection ordetection of the nonlinear optical light beam is one or more modesselected from the group consisting of reflection, transmission,evanescent wave, multiple internal reflection, near-field opticaltechniques, confocal, optical cavity, planar waveguide, fiber-optic anddielectric-slab waveguide, and near-field techniques.
 149. The method ofclaim 112, wherein a nonlinear-active label is covalently bound to saidtest molecule or to said one or more candidate binding partners. 150.The method of claim 112, wherein a nonlinear-active label is covalentlybound to a molecule that is noncovalently bound to said test molecule orto said one or more candidate binding partners.
 151. The method of claim112, which further comprises comparing the value of said one or morephysical properties measured in step (b) relative to the value of theone or more physical properties measured in the absence of said one ormore candidate binding partners.
 152. A method for detecting the degreeor extent of the conformational change induced by binding between a testmolecule and one or more candidate binding partners comprising: (a)contacting said test molecule with one or more candidate bindingpartners, wherein the test molecule or the one or more candidate bindingpartners is labeled with a nonlinear-active moiety that is not native tothe test molecule or the one or more candidate binding partners,respectively; (b) illuminating said contacted test molecule with one ormore light beams at one or more fundamental frequencies; and (c)measuring one or more physical properties of a nonlinear optical lightbeam emanating from said sample; wherein the extent of the change in thevalue of said one or more physical properties measured in step (c)relative to the value for said one or more physical properties measuredin the absence of said one or more candidate binding partners indicatesthe degree or extent of the conformational change that said bindinginduces.
 153. The method of claim 152, wherein the one or-more physicalproperties are intensities.
 154. The method of claim 152, wherein theone or more physical properties are polarization directions.
 155. Themethod of claim 152, wherein said test molecule is nonlinear-active inthe absence of an exogenous nonlinear-active label bound to the testmolecule.
 156. The method of claim 152, wherein said one or more lightbeams have a wavelength in the range of 10 to 10000 nanometers.
 157. Themethod of claim 152, which further comprises comparing the value of saidphysical properties measured in step (b) with the value of the physicalproperties measured in the absence of exposure to said one or morecandidate binding partners.
 158. The method of claim 157, wherein saidtest molecule or said candidate binding partners are bound to at leasttwo distinguishable nonlinear-active labels, wherein said one or morelight beams are multiple light beams, and step (b) comprises measuringone or more physical properties of at least two nonlinear optical lightbeams emanating from said sample.
 159. The method of claim 152, whereinsaid test molecule is purified.
 160. The method of claim 152, whereinsaid test molecule is a G protein-coupled receptor (GPCR).
 161. Themethod of claim 160, wherein said GPCR is bound to a nonlinear-activelabel.
 162. The method of any claims 152, wherein said nonlinear opticallight beam is second harmonic generated light.
 163. The method of claim152, which further comprises applying an electric field to said sample.164. The method of claim 163, wherein the electric field is stationaryin time, time-varying, or some combination thereof.
 165. The method ofclaim 152, wherein said sample further comprises a bulk phase.
 166. Themethod of claim 165, wherein an electric field is applied to said bulkphase.
 167. The method of claim 166, wherein the electric field isstationary in time, time-varying, or some combination thereof.
 168. Themethod of claim 152, wherein said one or more nonlinear optical lightbeams are second harmonics, third harmonics, sum frequencies ordifference frequencies of the one or more fundamental frequencies. 169.The method of claim 152, wherein said one or more candidate bindingpartners are patterned in an array format on a solid surface.
 170. Themethod of claim 169, wherein said one or more candidate binding partnerscomprise a plurality of different oligonucleotides or polynucleotides,said different oligonucleotides or polynucleotides each comprising adifferent sequence and attached to a different region on a solidsurface.
 171. The method of claim 169, wherein the oligonucleotides orpolynucleotides are patterned in a microarray format.
 172. The method ofclaim 152, wherein said one or more candidate binding partners compriseproteins patterned in an array format.
 173. The method of claim 152,wherein the step of illuminating involves reflection, transmission,evanescent wave, multiple internal reflection, near-field optics,confocal, optical cavity, planar waveguide, fiber-optic ordielectric-slab waveguide.
 174. The method of claim 152, wherein thestep of measuring involves reflection, transmission, evanescent wave,multiple internal reflection, near-field optics, confocal, opticalcavity, planar waveguide, fiber-optic or dielectric-slab waveguide. 175.The method of claim 152, wherein said one or more candidate bindingpartners are molecular beacon analogues.
 176. The method of claim 152,wherein said one or more candidate binding partners are attached to asurface.
 177. The method of claim 176, wherein said surface is a metalsurface, a semiconductor surface, a glass surface, a latex surface, agel substrate, a fiber-optic surface, a silica surface or a beadsurface.
 178. The method of claim 176, wherein said surface is anon-planar surface.
 179. The method of claim 176, wherein said surfaceis chemically derivatized.
 180. The method of claim 179, wherein saidsurface is derivatized with a self-assembled monolayer.
 181. The methodof claim 179, wherein said surface is derivatized with an organosilane.182. The method of claim 152, wherein the step of measuring is repeatedover different periods of time.
 183. The method of claim 152, whereinsaid one or more candidate binding partners is attached to aself-assembled monolayer.
 184. The method of claim 183, wherein theself-assembled monolayer is in the chemical family of silanes orterminal-functional silanes.
 185. The method of claim 152, wherein theone or more physical properties of the nonlinear optical light beammeasured indicate thermodynamic or kinetic properties of said binding.186. The method of claim 152, wherein said binding involves a-chemicalbond, an electrostatic force, physisorption, chemical affinity,chemisorption, molecular recognition, physico-chemical binding, hydrogenbond or hybridization process.
 187. The method of claim 152, wherein thesample further comprises one or more substances selected from the groupconsisting of decorator molecules, decorator particles, enhancers,modulators, inhibitors, molecular beacon analogues, and indicators. 188.The method of claim 152, wherein the mode of generation, collection ordetection of the nonlinear optical light beam is one or more modesselected from the group consisting of reflection, transmission,evanescent wave, multiple internal reflection, near-field opticaltechniques, confocal, optical cavity, planar waveguide, fiber-optic anddielectric-slab waveguide, and near-field techniques.
 189. The method ofclaim 152, wherein a nonlinear-active label is covalently bound to saidtest molecule or to said one or more candidate binding partners. 190.The method of claim 152, wherein a nonlinear-active label is covalentlybound to a molecule that is noncovalently bound to said test molecule orto said one or more candidate binding partners.
 191. The method of claim152, which further comprises comparing the value of said one or morephysical properties measured in step (b) relative to the value of saidone or more physical properties measured in the absence of said one ormore candidate binding partners.
 192. The method of claims 1, whereinsaid one or more candidate binding partners are not attached to asurface.
 193. The method of claims 75, wherein said one or morecandidate binding partners are not attached to a surface.
 194. Themethod of claims 112, wherein said one or more candidate bindingpartners are not attached to a surface.
 195. The method of claims 152,wherein said one or more candidate binding partners are not attached toa surface.